Neoplasm specific antibodies and uses thereof

ABSTRACT

The present invention features polypeptides, such as antibodies, and their use in the treatment and diagnosis of neoplasms.

BACKGROUND OF THE INVENTION

The present invention is related to the field of cancer diagnosis andtreatment and, more specifically, to the identification of polypeptides,such as antibodies, useful in the diagnosis, detection, monitoring, andtreatment of neoplasms in a mammal, e.g., a human.

In the United States well over one million individuals are diagnosedwith cancer each year. Although recent advances in the medical fieldhave significantly improved the rate of survival among cancer patients,a large number of cancer-related deaths still could be prevented by theearly diagnosis of the tumor. Accordingly, at the time of initialdiagnosis, an alarming number of patients have already reached latestages of the disease.

With respect to colorectal cancer, the prognosis is usually poor in 50%of all cases because the tumor is often undetected until the disease hasspread and reached a terminal stage. Similarly, approximately 75% ofwomen are diagnosed with ovarian cancer after the disease has alreadyreached an advanced stage (stage III or IV) because the symptoms ofovarian cancer are often vague or “silent.” Despite aggressive surgicalintervention and new chemotherapeutic regimens, the overall 5-yearsurvival rate for these women with advanced stage ovarian cancer hasremained constant over the past 30 years, at approximately 15%.Conversely, women diagnosed with cancer confined to the ovary (stage I)have an overall 5-year survival rate approaching 90%.

Clearly, there is a need for the early and improved detection andtreatment of neoplasms (e.g., stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary,adenocarcinoma of the endometrium, or adenocarcinoma of the uterus), asthis would increase the chance of treating the neoplasm and, thereby,lead to an improved prognosis for long-term survival.

SUMMARY OF THE INVENTION

We have discovered a class of polypeptides which react with an epitopespecific for neoplastic cells. These polypeptides are not only excellentdiagnostic tools, but also can induce apoptosis of the neoplastic cellsto which they bind. This latter characteristic results in a treatmentfor neoplastic diseases that lacks the side-effects of many existingtherapeutics.

The present invention features polypeptides, such as monoclonalantibodies that may be used in the diagnosis and treatment of aneoplasm. Accordingly, in the first aspect, the invention features apurified polypeptide that induces apoptosis of a neoplastic cell towhich it binds, but does not induce apoptosis of a non-neoplastic cell,where the antibody specifically binds to at least one of HT-29 (ATCCAccession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCCAccession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), or BXPC-3 (ATCC Accession No. CRL-1687)cells, and not to non-neoplastic cells.

In a second aspect, the invention features a purified polypeptide thatinduces apoptosis of a neoplastic cell to which it binds, but does notinduce apoptosis of a non-neoplastic cell, where the polypeptidespecifically binds to a stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary,adenocarcinoma of the endometrium, or adenocarcinoma of the uterus celland not to a non-neoplastic cell.

In the third aspect, the invention features a purified polypeptide thatinhibits cell proliferation when bound to a neoplastic cell, but doesnot inhibit cell proliferation of a non-neoplastic cell, where thepolypeptide specifically binds to at least one of HT-29 (ATCC AccessionNo. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCC Accession No.HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZ Accession No. ACC144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1 (ATCC Accession No.CRL-1682), or BXPC-3 (ATCC Accession No. CRL-1687) cells, and not tonon-neoplastic cells.

In a desirable embodiment of the first and third aspects of theinvention, the polypeptide binds to ASPC-1 (ATCC Accession No. CRL-1682)and BXPC-3 (ATCC Accession No. CRL-1687) cells and not to non-neoplasticcells, and the neoplastic cell is a stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary, oradenocarcinoma of the uterus cell. In addition, the polypeptide mayinclude the sequence of SEQ ID NO:1 or 3, or a sequence that issubstantially identical to the sequence of SEQ ID NO:1 or 3.Furthermore, the polypeptide also may be produced by the PM-1 cell linedeposited at the DSMZ under Accession No. DSM ACC2599.

In another desirable embodiment of the first and third aspects, thepolypeptide binds to HT-29 (ATCC Accession No. HTB-38; DSMZ AccessionNo. ACC 299), CACO-2 (ATCC Accession No. HBT-37; DSMZ Accession No.

ACC 169), COLO-320 (DSMZ Accession No. ACC 144), COLO-206F (DSMZAccession No. ACC 21), ASPC-1 (ATCC Accession No. CRL-1682), and BXPC-3(ATCC Accession No. CRL-1687) cells and not to non-neoplastic cells, andthe neoplastic cell is a stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary, oradenocarcinoma of the uterus cell. In addition, the polypeptide mayinclude the sequence of SEQ ID NO:5 or 7, or a sequence that issubstantially identical to the sequence of SEQ ID NO:5 or 7.Furthermore, the polypeptide also may be produced by the PM-2 cell linedeposited at the DSMZ under Accession No. DSM ACC2600.

In a further desirable embodiment of the first and third aspects, thepolypeptide binds to CACO-2 (ATCC Accession No. BBT-37; DSMZ AccessionNo. ACC 169) and COLO-206F (DSMZ Accession No. ACC 21) cells and not tonon-neoplastic cells, and the neoplastic cell is a colorectaladenocarcinoma or adenocarcinoma of the endometrium cell. In addition,the polypeptide may include the sequence of SEQ ID NO:9 or 11, or asequence that is substantially identical to the sequence of SEQ ID NO:9or 11. Furthermore, the polypeptide also may be produced by the CM-2cell line deposited at the DSMZ under Accession No. DSM ACC2598.

The fourth aspect of the invention features a purified polypeptide thatinhibits cell proliferation when bound to a neoplastic cell, but doesnot inhibit cell proliferation of a non-neoplastic cell, where thepolypeptide specifically binds to a stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary,adenocarcinoma of the endometrium, or adenocarcinoma of the uterus celland not to a non-neoplastic cell.

In desirable embodiments of the first four aspects of the invention, thepolypeptide includes an antibody or a functional fragment thereof. Forexample, the functional fragment may be selected from the groupconsisting of V_(L), V_(H), F_(V), F_(C), Fab, Fab′, and F(ab′)₂. Inaddition, the functional fragment may include a fragment that issubstantially identical to the sequence of SEQ ID NOS:1, 3, 5, 7, 9, or11 or may include a fragment of the sequence of SEQ ID NO:1, 3, 5, 7, 9,or 11. In addition, the fragment may be substantially identical to apolypeptide including amino acids 26-31, 49-51, and 88-95 of SEQ ID NO:1or amino acids 11-18, 36-43, and 82-90 of SEQ ID NO:3; a polypeptideincluding amino acids 26-34, 52-58, and 97-103 of SEQ ID NO:5 or aminoacids 11-18, 36-43, and 82-100 of SEQ ID NO:7; or a polypeptideincluding amino acids 26-34, 51-54, and 91-99 of SEQ ID NO:9 or aminoacids 16-22, 40-47, and 86-100 of SEQ ID NO:11.

The fifth aspect of the invention features a purified polypeptide thatincludes the amino acid sequence of SEQ ID NO:1; the sixth aspect of theinvention features a purified polypeptide that includes the amino acidsequence of SEQ ID NO:3; the seventh aspect of the invention features apurified polypeptide that includes the amino acid sequence of SEQ IDNO:5; the eighth aspect of the invention features a purified polypeptidethat includes the amino acid sequence of SEQ ID NO:7; the ninth aspectof the invention features a purified polypeptide that includes the aminoacid sequence of SEQ ID NO:9; and the tenth aspect of the inventionfeatures a purified polypeptide that includes the amino acid sequence ofSEQ ID NO:11.

Furthermore, the invention features a purified polypeptide includingamino acid 26-31, 49-51, and 88-95 of SEQ ID NO:1, a purifiedpolypeptide including amino acids 11-18, 36-43, and 82-90 of SEQ IDNO:3, a purified polypeptide including amino acids 26-34, 52-58, and97-103 of SEQ ID NO:5, a purified polypeptide including amino acids11-18, 36-43, and 82-100 of SEQ ID NO:7, a purified polypeptideincluding amino acids 26-34, 51-54, and 91-99 of SEQ ID NO:9, and apurified polypeptide including amino acids 16-22, 40-47, and 86-100 ofSEQ ID NO:11. Moreover, the invention features a purified polypeptideincluding amino acid 26-31, 49-51, and 88-95 of SEQ ID NO:1 and aminoacids 11-18, 36-43, and 82-90 of SEQ ID NO:3, a purified polypeptideincluding amino acids 26-34, 52-58, and 97-103 of SEQ ID NO:5 and aminoacids 11-18, 36-43, and 82-100 of SEQ ID NO:7, and a purifiedpolypeptide including amino acids 26-34, 51-54, and 91-99 of SEQ ID NO:9and amino acids 16-22, 40-47, and 86-100 of SEQ ID NO:11.

In an eleventh aspect, the invention features a purified polypeptidethat includes the amino acid sequence of SEQ ID NOS:1 and 3; in atwelfth aspect, the invention features a purified polypeptide thatincludes the amino acid sequence of SEQ ID NOS:5 and 7; and in athirteenth aspect, the invention features a purified polypeptide thatincludes the amino acid sequence of SEQ ID NOS:9 and 11.

In a desirable embodiment of the first thirteen aspects of theinvention, the polypeptide is an antibody, such as a monoclonalantibody, e.g., a human monoclonal antibody.

In a fourteenth aspect, the invention features a cell that expresses thepolypeptide of the first or second aspect and in a fifteenth aspect, theinvention features a cell that expresses the polypeptide of the third orfourth aspect of the invention. In the sixteenth aspect, the inventionfeatures a cell that expresses a polypeptide that includes a sequencethat is substantially identical to the amino acid sequence of SEQ IDNO:1 or 3, and in desirable embodiments of this aspect, the polypeptideincludes the sequence of SEQ ID NO:1 or 3, or both SEQ ID NO:1 and 3.

In a seventeenth aspect, the invention features a cell that expresses apolypeptide that includes a sequence that is substantially identical tothe amino acid sequence of SEQ ID NO:5 or 7, and in desirableembodiments of this aspect, the polypeptide includes the sequence of SEQID NO:5 or 7, or both SEQ ID NO:5 and 7.

In an eighteenth aspect, the invention features a cell that expresses apolypeptide that includes a sequence that is substantially identical tothe amino acid sequence of SEQ ID NO:9 or 11, and in desirableembodiments of this aspect, the polypeptide includes the sequence of SEQID NO:9 or 11, or both SEQ ID NO:9 and 11. In further desirableembodiments of the fourteenth through eighteenth aspects of theinvention, the cell is a hybridoma.

In the nineteenth aspect, the invention features a method of generatingthe cell of the fourteenth aspect. This method involves the steps of:(a) contacting lymphocytes with a heteromyeloma cell line underconditions that result in the fusion of a lymphocyte with aheteromyeloma cell, where the fusion results in a hybridoma, (b)determining whether the hybridoma produces a polypeptide that inducesapoptosis of a neoplastic cell to which it binds, but does not induceapoptosis of a non-neoplastic cell, and (c) determining whether thehybridoma produces a polypeptide that specifically binds to at least oneof HT-29 (ATCC Accession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2(ATCC Accession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), or BXPC-3 (ATCC Accession No. CRL-1687)cells and not to non-neoplastic cells.

In the twentieth aspect, the invention features a method of generatingthe cell of the fifteenth aspect. This method involves the steps of: (a)contacting lymphocytes with a heteromyeloma cell line under conditionsthat result in the fusion of a lymphocyte with a heteromyeloma cell,where the fusion results in a hybridoma, (b) determining whether thehybridoma produces a polypeptide that inhibits proliferation in aneoplastic cell to which it binds, but does not inhibit proliferation ina non-neoplastic cell, and (c) determining whether the hybridomaproduces a polypeptide that specifically binds to at least one of HT-29(ATCC Accession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCCAccession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), or BXPC-3 (ATCC Accession No. CRL-1687)cells and not to non-neoplastic cells.

In a twenty-first aspect, the invention features a use of the purifiedpolypeptide of any one of the first thirteen aspects of the invention ina method of diagnosing a neoplasm in a mammal, e.g., a human. Thismethod involves the steps of: (a) contacting a cell or tissue sample ofthe mammal with the purified polypeptide of any one of the firstthirteen aspects of the invention, and (b) detecting whether thepurified polypeptide binds to the cell or tissue sample, where bindingof the purified polypeptide to the cell or tissue sample is indicativeof the mammal having a neoplasm.

In desirable embodiments of the twenty-first aspect of the invention,the neoplasm is a stomach adenocarcinoma, colorectal adenocarcinoma,squamous cell lung carcinoma, lung adenocarcinoma, squamous cellcarcinoma of the esophagus, adenocarcinoma of the pancreas, urothelcarcinoma of the urinary bladder, renal cell carcinoma of the kidney,adenocarcinoma of the prostate, ductal carcinoma of the breast, lobularcarcinoma of the breast, adenocarcinoma of the ovary, adenocarcinoma ofthe endometrium, or adenocarcinoma of the uterus. In further desirableembodiments of this aspect, the polypeptide is an antibody or thepolypeptide is conjugated to a detectable agent selected from the groupconsisting of a radionuclide, a fluorescent marker, an enzyme, acytotoxin, a cytokine, and a growth inhibitor. Further, the polypeptidemay be conjugated to a protein purification tag, e.g., a cleavableprotein purification tag.

In the twenty-second aspect, the invention features a use of thepurified polypeptide of any one of the first thirteen aspects of theinvention in a method of treating a proliferative disorder in a mammal,e.g., a human. This method involves the step of contacting a cell ortissue sample with the purified polypeptide of any one of the firstthirteen aspects, where binding of the purified polypeptide to the cellor tissue sample results in the induction of apoptosis of the cell ortissue sample.

In desirable embodiments of the twenty-second aspect of the invention,the proliferative disorder is a stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary,adenocarcinoma of the endometrium, or adenocarcinoma of the uterus. Infurther desirable embodiments of this aspect, the polypeptide is anantibody or the polypeptide is conjugated to a detectable agent selectedfrom the group consisting of a radionuclide, a fluorescent marker, anenzyme, a cytotoxin, a cytokine, and a growth inhibitor. Desirably, thedetectable agent is capable of inducing apoptosis of the cell or tissuesample. In addition, the polypeptide may be conjugated to a proteinpurification tag, e.g., a protein purification tag that is cleavable.

In the twenty-third aspect, the invention features a use of the purifiedpolypeptide of any one of the first thirteen aspects of the invention ina method of treating a proliferative disorder in a mammal, e.g., ahuman. This method involves the step of contacting a cell or tissuesample with the purified polypeptide of any one of the first thirteenaspects of the invention, where binding of the purified polypeptide tothe cell or tissue sample results in a reduction in proliferation of thecell or of a cell in the tissue sample.

In desirable embodiments of the twenty-third aspect of the invention,the proliferative disorder is a stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary,adenocarcinoma of the endometrium, or adenocarcinoma of the uterus. Infurther desirable embodiments of this aspect, the polypeptide is anantibody or the polypeptide is conjugated to a detectable agent selectedfrom the group consisting of a radionuclide, a fluorescent marker, anenzyme, a cytotoxin, a cytokine, and a growth inhibitor. Desirably, thedetectable agent is capable of inhibiting cell proliferation of the cellor tissue sample. In addition, the polypeptide may be conjugated to aprotein purification tag, e.g., a protein purification tag that iscleavable.

In a twenty-fourth aspect, the invention features a medicament thatcontains the purified polypeptide of any one of the first thirteenaspects of the invention in a pharmaceutically acceptable carrier.

In the twenty-fifth aspect, the invention features a diagnostic agentthat contains the purified polypeptide of any one of the first thirteenaspects of the invention.

In the twenty-sixth aspect, the invention features an isolated nucleicacid molecule that is substantially identical to the sequence of SEQ IDNO:2, 4, 6, 8, 10, or 12, and in the twenty-seventh aspect, theinvention features an isolated nucleic acid sequence that hybridizesunder high stringency conditions to the sequence of SEQ ID NO:2, 4, 6,8, 10, or 12.

In the twenty-eighth aspect, the invention features an isolated nucleicacid molecule that includes the sequence of SEQ ID NO:2, 4, 6, 8, 10, or12; in the twenty-ninth aspect, the invention features an isolatednucleic acid molecule that includes sequences that are substantiallyidentical to nucleotides 76-93, 145-153, and 262-285 of SEQ ID NO:2. Ina desirable embodiment of this aspect, the isolated nucleic acidmolecule includes nucleotides 76-93, 145-153, and 262-285 of SEQ IDNO:2.

The thirtieth aspect of the invention features an isolated nucleic acidmolecule that includes sequences that are substantially identical tonucleotides 31-54, 106-129, and 244-270 of SEQ ID NO:4. In a desirableembodiment of this aspect, the isolated nucleic acid molecule includesnucleotides 31-54, 106-129, and 244-270 of SEQ ID NO:4

In the thirty-first aspect, the invention features an isolated nucleicacid molecule that includes sequences that are substantially identicalto nucleotides 76-102, 154-174, and 289-309 of SEQ ID NO:6. In adesirable embodiment of this aspect, the isolated nucleic acid moleculeincludes nucleotides 76-102, 154-174, and 289-309 of SEQ ID NO:6.

In the thirty-second aspect, the invention features an isolated nucleicacid molecule that includes sequences that are substantially identicalto nucleotides 31-54, 106-129, and 244-300 of SEQ ID NO:8. In adesirable embodiment of this aspect, the isolated nucleic acid moleculeincludes nucleotides 31-54, 106-129, and 244-300 of SEQ ID NO:8

In the thirty-third aspect, the invention features an isolated nucleicacid molecule that includes sequences that are substantially identicalto nucleotides 76-102, 151-162, and 271-297 of SEQ ID NO:10. In adesirable embodiment of this aspect, the isolated nucleic acid moleculeincludes nucleotides 76-102, 151-162, and 271-297 of SEQ ID NO:10.

In the thirty-fourth aspect, the invention features an isolated nucleicacid molecule that includes sequences that are substantially identicalto nucleotides 46-66, 118-141, and 256-300 of SEQ ID NO:12. In adesirable embodiment of this aspect, the isolated nucleic acid moleculeincludes nucleotides 46-66, 118-141, and 256-300 of SEQ ID NO:12.

In the thirty-fifth aspect, the invention features a vector includingthe nucleic acid molecule of any one of that twenty-sixth throughthirty-fourth aspects of the invention, and in the thirty-sixth aspect,the invention features a cell that includes the vector of thethirty-fifth aspect.

The thirty-seventh aspect of the invention features a method ofpreparing the purified polypeptide of the first four aspects of theinvention. This method involves contacting a cell with the vector of thethirty-fifth aspect of the invention and isolating the polypeptideexpressed by the cell.

Definitions

By “detectable agent” is meant a compound that is linked to a diagnosticagent to facilitate detection. Such a “detectable agent” may becovalently or non-covalently linked to a diagnostic agent. In addition,the linkage may be direct or indirect. Examples of “detectable agents”include, protein purification tags, cytotoxins, enzymes, paramagneticlabels, enzyme substrates, co-factors, enzymatic inhibitors, dyes,radionuclides, chemiluminescent labels, fluorescent markers, growthinhibitors, cytokines, antibodies, and biotin.

By a “diagnostic agent” is meant a compound that may be used to detect aneoplastic cell by employing any one of the assays described herein aswell as any other method that is standard in the art. A diagnostic agentmay include, for example, an antibody which specifically binds to atleast one of the following cells: HT-29 (ATCC Accession No. HTB-38; DSMZAccession No. ACC 299), CACO-2 (ATCC Accession No. HBT-37; DSMZAccession No. ACC 169), COLO-320 (DSMZ Accession No. ACC 144), COLO-206F(DSMZ Accession No. ACC 21), ASPC-1 (ATCC Accession No. CRL-1682), andBXPC-3 (ATCC Accession No. CRL-1687), but not to non-neoplastic cells.In addition, a “diagnostic agent” may inhibit cell proliferation, induceapoptosis, or both only when it is bound to a neoplastic cell, but not anon-neoplastic cell.

Examples of neoplastic cells that may be detected with such a“diagnostic agent” include stomach adenocarcinoma, colorectaladenocarcinoma, squamous cell lung carcinoma, lung adenocarcinoma,squamous cell carcinoma of the esophagus, adenocarcinoma of thepancreas, urothel carcinoma of the urinary bladder, renal cell carcinomaof the kidney, adenocarcinoma of the prostate, ductal carcinoma of thebreast, lobular carcinoma of the breast, adenocarcinoma of the ovary,adenocarcinoma of the endometrium, or adenocarcinoma of the uteruscells. Moreover, a “diagnostic agent” may include, for example,peptides, polypeptides, synthetic organic molecules, naturally-occurringorganic molecules, nucleic acid molecules, and components thereof, aswell as one or more detectable agent covalently or non-covalently linkedto the diagnostic agent.

By a “functional fragment,” as used herein in reference to polypeptide,is meant a fragment that retains at least one biological activity of thefull-length polypeptide. Examples of such a biological activity are theability to specifically bind an antigen, induce apoptosis, and/orinhibit cell proliferation. These biological activities may bedetermined, for example, using any one of the assays described herein.

Examples of functional fragments of an antibody are V_(L), V_(H), F_(V),F_(C), Fab, Fab′, or F(ab′)₂ fragments (see, e.g., Huston et al., CellBiophys. 22:189-224, 1993; and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988). Desirably, a “functionalfragment” has an amino acid sequence that is substantially identical toa fragment, e.g., 3, 4, 5, 10, 15, 20, 15, 30, 50, 75, or 100 contiguousamino acids, of the amino acid sequence of SEQ ID NO:1, 3, 5, 7, 9, or11. In more desirable embodiments, a “functional fragment” is identicalto a fragment of the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11. Such a“functional fragment” may contain 3, 4, 5, 10, 15, 20, 15, 30, 50, 75,or 100 contiguous amino acids of SEQ ID NO: 1, 3, 5, 7, 9, or 11, or maybe the entire amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11. Indesirable embodiments, such a fragment includes one or more of theComplement Determining Regions (CDR) of the V_(H) or the V_(L) regionsof the PM-1, PM-2, or CM-2 antibody. For example, a functional fragmentmay include amino acids 26-31, 49-51, and/or 88-95 of SEQ ID NO:1; aminoacids 11-18, 36-43 and/or 82-90 of SEQ ID NO:3; amino acids 26-34,52-58, and/or 97-103 of SEQ ID NO:5; amino acids 11-18, 36-43, and/or82-100 of SEQ ID NO:7; amino acids 26-34, 51-54, and/or 91-99 of SEQ IDNO:9; or amino acids 16-22, 40-47, and/or 86-100 of SEQ ID NO:11.

By “high stringency hybridization conditions” is meant, for example,hybridization at approximately 42° C. in about 50% formamide, 0.1 mg/mlsheared salmon sperm DNA, 1% SDS, 2× SSC, 10% Dextran Sulfate, a firstwash at approximately 65° C. in about 2×SSC, 1% SDS, followed by asecond wash at approximately 65° C. in about 0.1×SSC. Alternatively,“high stringency hybridization conditions” may include hybridization atapproximately 42° C. in about 50% formamide, 0.1 mg/ml sheared salmonsperm DNA, 0.5% SDS, 5×SSPE, 1× Denhardt's, followed by two washes atroom temperature in 2×SSC, 0.1% SDS, and two washes at between 55-60° C.in 0.2×SSC, 0.1% SDS.

A “hybridoma,” as used herein, is any cell that is artificially createdby the fusion of a normal cell such as an activated lymphocyte with aneoplastic cell, e.g., a myeloma. The hybrid cell, which results fromthe fusion of at least two cells, may produce a monoclonal antibody or Tcell product identical to those produced by theimmunologically-competent parent. In addition, these cells, like theneoplastic parent, are immortal.

“Inhibiting cell proliferation,” as used herein, refers to a reductionin the rate of cell division of a cell in comparison with the normalrate of cell division of that type of cell. Inhibition of cellproliferation may be assayed using a number of methods standard in theart, for example, the MTT cell proliferation assay described herein,BrdU incorporation, and ³H thymidine uptake. Such assays are described,for example, in Ausubel et al., Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001; and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989.Desirably, the inhibition of cell proliferation is 20%, 40%, 50%, or75%. In desirable embodiments, the inhibition of cell proliferation is80%, 90%, 95%, or even a complete inhibition of cell proliferation.

“Inducing apoptosis,” as used herein, refers to the appearance ofcharacteristics in a cell that are well defined in the art (see, e.g.,Wyllie et al., Br. J. Cancer 80 Suppl. 1:34-37, 1999; Kerr et al., Br.J. Cancer 26:239-257, 1972). These characteristics include morphologicalcharacteristics, such as membrane blebbing, DNA condensation, as well aschanges in F-actin content, mitochondrial mass, and membrane potential.The induction of apoptosis may be assayed using a number of methodsstandard in the art, for example, a cell death ELISA, TUNEL staining,DNA stains, e.g., Hoechst 33258, and staining with various vital dyessuch as acridine orange, Mito Tracker Red® staining (Molecular Probes,Eugene, Oreg.), and Annexin V® staining (Becton Dickinson, NJ). As usedherein “inducing apoptosis” refers to an increase in the number of cellsundergoing apoptosis when compared with a control cell population. Forinstance, the increase of apoptosis may be 10%, 20%, 40%, 50%, or 75%.In desirable embodiments, the induction of apoptosis results in anincrease of apoptosis that is 2-fold, 3-fold, 10-fold, or even 100-foldover that seen in a control cell population.

A “neoplastic cell,” as used herein, refers to a cell which isundergoing cell division, not undergoing apoptosis, or both, underinappropriate conditions. For example, a “neoplastic cell” may undergocell division when a corresponding non-neoplastic cell does not undergocell division, or, alternatively, a “neoplastic cell” may not respond tonormal cell-cycle checkpoint controls.

A “proliferative disease,” as used herein, refers to any disorder thatresults in the abnormal proliferation of a cell. Specific examples ofproliferative diseases are various types of neoplasms, such as stomachadenocarcinoma, colorectal adenocarcinoma, squamous cell lung carcinoma,lung adenocarcinoma, squamous cell carcinoma of the esophagus,adenocarcinoma of the pancreas, urothel carcinoma of the urinarybladder, renal cell carcinoma of the kidney, adenocarcinoma of theprostate, ductal carcinoma of the breast, lobular carcinoma of thebreast, adenocarcinoma of the ovary, adenocarcinoma of the endometrium,or adenocarcinoma of the uterus. However, proliferative diseases mayalso be the result of the cell becoming infected with a transformingvirus.

A “protein purification tag,” as used herein, is a peptide, e.g., anepitope tag, that is covalently or non-covalently added to a protein toaid in the purification of the protein. Desirably such peptides bindwith high affinity to an antibody or to another peptide such as biotinor avidin. Commercially available examples of epitope tags includeHis-tags, HA-tags, FLAG®-tags, and c-Myc-tags. However, any epitope thatis recognized by an antibody also may be used as a protein purificationtag. See, for example, Ausubel et al., Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., (1989). Protein purification tags may be cleaved from a protein,for example, by using an enzyme, e.g., thrombin, or a chemical, e.g.,cyanogen bromide.

By “specifically recognize,” as used herein in reference to apolypeptide, e.g., an antibody, is meant an increased affinity of apolypeptide for a particular protein, e.g., an antigen, relative to anequal amount of any other protein. For example, an antibody, e.g., thePM-1, PM-2, or CM-2 human monoclonal antibody, that specifically bindsto HT-29 (American Type Culture Collection (“ATCC”) Accession No.HTB-38, Gerthan Collection of Microorganisms and Cell Cultures (“DSMZ”)Accession No. ACC 299), CACO-2 (ATCC Accession No. HBT-37, DSMZAccession No. ACC 169), COLO-320 (DSMZ Accession No. ACC 144), COLO-206F(DSMZ Accession No. ACC 21), ASPC-1 (ATCC Accession No. CRL-1682), orBXPC-3 (ATCC Accession No. CRL-1687) cells desirably has an affinity forits antigen that is least 2-fold, 5-fold, 10-fold, 30-fold, or 100-foldgreater than for an equal amount of any other antigen, including relatedantigens. Binding of a polypeptide to another polypeptide may bedetermined as described herein, and by any number of standard methods inthe art, e.g., Western analysis, ELISA, or co-immunoprecipitation.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 50%, 75%, 80%, 85%, or 90% identity to a referenceamino acid (e.g., the sequence of SEQ ID NO:1, 3, 5, 7, 9, or 11) ornucleic acid sequence (e.g., the sequence of SEQ ID NO:2, 4, 6, 8, 10,or 12), or a fragment thereof. In desirable embodiments, the polypeptideor nucleic acid sequence is at least 95%, 98%, 99%, or even 100%identical to a reference amino acid or nucleic acid sequence. Forpolypeptides, the length of comparison sequences will generally be atleast 3, 4, 5, 6, 8, 10, or 15 amino acids and desirably at least 20 or25 contiguous amino acids. In more desirable embodiments, the length ofcomparison sequences is at least 30, 50, 75, 90, 95, or 100 contiguousamino acids, or even the full-length amino acid sequence. For nucleicacids, the length of comparison sequences will generally be at least 9,10, 15, 20, or 25 contiguous nucleotides, and desirably at least 30contiguous nucleotides. In more desirable embodiments, the length ofcomparison sequences is at least 50, 75, 150, 225, 270, 285, or 300contiguous nucleotides, or even the full-length nucleotide sequence.

Sequence identity may be measured using sequence analysis software onthe default setting (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Such software may matchsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

Multiple sequences may also be aligned using the Clustal W(1.4) program(produced by Julie D. Thompson and Toby Gibson of the European MolecularBiology Laboratory, Germany and Desmond Higgins of EuropeanBioinformatics Institute, Cambridge, UK) by setting the pairwisealignment mode to “slow,” the pairwise alignment parameters to includean open gap penalty of 10.0 and an extend gap penalty of 0.1, as well assetting the similarity matrix to “blosum.” In addition, the multiplealignment parameters may include an open gap penalty of 10.0, an extendgap penalty of 0.1, as well as setting the similarity matrix to“blosum,” the delay divergent to 40%, and the gap distance to 8.

By “purified” or “isolated” is meant separated from other componentsthat naturally accompany it. Typically, a factor is substantially purewhen it is at least 50%, by weight, free from proteins, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated, or in reference to a nucleic acid molecule, is free from thenucleic acid sequences that naturally flank the sequence of the nucleicacid molecule in the genome of an organism. Desirably, the factor is atleast 75%, more desirably, at least 90%, and most desirably, at least99%, by weight, pure. A substantially pure factor may be obtained bychemical synthesis, separation of the factor from natural sources, orproduction of the factor in a recombinant host cell that does notnaturally produce the factor. Proteins, vesicles, and organelles may bepurified by one skilled in the art using standard techniques, such asthose described by Ausubel et al. (Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001). The factor is desirably atleast 2, 5, or 10 times as pure as the starting material, as measuredusing polyacrylamide gel electrophoresis, column chromatography, opticaldensity, HPLC analysis, or Western analysis (Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience, New York, 2001).Desirable methods of purification include immunoprecipitation, columnchromatography such as immunoaffinity chromatography and nickel affinitycolumns, magnetic bead immunoaffinity purification, and panning with aplate-bound antibody.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the Drawings, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a series of images showing immunohistochemical stainingof PM-1 and PM-2 on different carcinomas. Paraffin sections were stainedwith hematoxylin-eosin, positive control antibodies (CK7 foradenocarcinoma of the pancreas, CK5/6 for squamous cell carcinoma of thelung, and CK8 for invasive ductal adenocarcinoma of the breast),secondary antibody alone as a negative control, and either antibody PM-1or PM-2. FIG. 1A shows staining of an adenocarcinoma of the pancreas;FIG. 1B shows staining of a squamous cell carcinoma of the lung; FIG. 1Cshows staining of an adenocarcinoma of the pancreas; and FIG. 1D showsstaining of an invasive ductal carcinoma of the breast. The originalmagnification for these images was 200×.

FIG. 2 is a series of images showing immunohistochemical staining ofCM-2 of an adenocarcinoma of the colon. Paraffin sections were stainedwith hematoxylin-eosin, positive control antibody (AE1/AE3 foradenocarcinoma of the colon), secondary antibody alone as a negativecontrol, and antibody CM-2. The original magnification for these imageswas 200×.

FIGS. 3A and 3B are a series of graphs depicting the functional analysisof antibodies PM-1, PM-2, and CM-2 in vitro. The consequences ofantibody treatment on the proliferation of different carcinoma celllines were measured using an MTT proliferation assay. FIG. 3A shows theconcentration dependent inhibition of cell proliferation with antibodiesPM-1 and PM-2 on pancreas carcinoma cell line BXPC-3. FIG. 3B shows theconcentration dependent inhibition of cell proliferation with antibodyCM-2 on colon carcinoma cell line Colo-206F. The control for theseexperiments was depleted cell culture supernatant with an unrelated IgMantibodies added at similar concentrations.

FIGS. 4A and 4B are a series of graphs showing that the PM-1, PM-2, andCM-2 antibodies induce apoptosis. In these experiments, apoptosis wasdetected using the Cell Death Detection ELISA^(PLUS) apoptosis assay.FIG. 4A shows PM-1 and PM-2 monoclonal antibody-induced apoptosis ofpancreas adenocarcinoma cell line BXPC-3. FIG. 4B shows CM-2 monoclonalantibody-induced apoptosis of Colon adenocarcinoma cell line CACO-2. Thecontrol in these experiments was depleted cell culture supernatant at asimilar concentration.

FIGS. 5A-5C is a series of Western blots showing the proteins recognizedby human monoclonal antibodies PM-1, PM-2, and CM-2 on membrane extractsof carcinoma cell lines. FIG. 5A shows a Western blot of a membraneextract of pancreas carcinoma cell line BXPC-3. Here, antibody PM-1recognizes two main bands of about 35 and 65 kDa. FIG. 5B shows aWestern blot of the same cell line as used in FIG. 5A, but here the blotwas incubated with the PM-2 antibody. The PM-2 antibody recognizes twobands with molecular weights of approximately 55 and 115 kDa. FIG. 5Cshows a Western blot of membrane extracts of colon carcinoma cell lineCACO2 incubated with the CM-2 antibody. The CM-2 antibody reacts withproteins in the range of 40 to 50 kDa. As a control in theseexperiments, unrelated human IgM was added at a similar concentration torule out non-specific binding.

FIGS. 6A and 6B are a series of graphs of the results of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)reduction assays for mitochondrial dehydrogenase activity showing thatthe PM-1 monoclonal antibody inhibits cell proliferation and decreasessurvival, or induces apoptosis of BXPC-3 pancreatic carcinoma cellsafter 24 hours of incubation (FIG. 6A) and after 48 hours of incubation(FIG. 6B).

FIG. 7 is a graph of the results of a cell death enzyme-linkedimmunosorbent assay (ELISA) showing that the PM-1 monoclonal antibodyinduces apoptosis of BXPC-3 cells after 24 hours of incubation.

FIGS. 8A and 8B are a series of graphs of the MTT reduction assays formitochondrial dehydrogenase activity showing that the PM-2 monoclonalantibody inhibits cell proliferation and decreases survival, or inducesapoptosis of BXPC-3 pancreatic carcinoma cells after 24 hours ofincubation (FIG. 8A) and after 48 hours of incubation (FIG. 8B).

FIG. 9 is a graph of the results of an ELISA showing that the PM-2monoclonal antibody induces apoptosis of BXPC-3 cells after 24 hours ofincubation.

FIGS. 10A and 10B are a series of graphs of the results of MTT reductionassays for mitochondrial dehydrogenase activity showing that the CM-2monoclonal antibody inhibits cell proliferation and decreases survival,or induces apoptosis of COLO-206F colon carcinoma cells after 24 hoursof incubation (FIG. 10A) and after 48 hours of incubation (FIG. 10B).

FIG. 11 is a graph of the results of an ELISA showing that the CM-2monoclonal antibody induces apoptosis of CACO-2 cells after 24 hours ofincubation.

FIG. 12 is the amino acid sequence (SEQ ID NO:1) and the nucleic acidsequence (SEQ ID NO:2) of the variable region of the light chain ofhuman monoclonal antibody PM-1. Complement Determining Regions (CDR) 1-3also are shown.

FIG. 13 is the amino acid sequence (SEQ ID NO:3) and the nucleic acidsequence (SEQ ID NO:4) of the variable region of the heavy chain ofhuman monoclonal antibody PM-1. CDR1-3 also are shown.

FIG. 14 is the amino acid sequence (SEQ ID NO:5) and the nucleic acidsequence (SEQ ID NO:6) of the variable region of the light chain ofhuman monoclonal antibody PM-2. CDR1-3 also are shown.

FIG. 15 is the amino acid sequence (SEQ ID NO:7) and the nucleic acidsequence (SEQ ID NO:8) of the variable region of the heavy chain ofhuman monoclonal antibody PM-2. CDR1-3 also are shown.

FIG. 16 is the amino acid sequence (SEQ ID NO:9) and the nucleic acidsequence (SEQ ID NO:10) of the variable region of the light chain ofhuman monoclonal antibody CM-2. CDR1-3 also are shown.

FIG. 17 is the amino acid sequence (SEQ ID NO:11) and the nucleic acidsequence (SEQ ID NO:12) of the variable region of the heavy chain ofhuman monoclonal antibody CM-2. CDR1-3 also are shown.

DETAILED DESCRIPTION

The present invention features polypeptides, such as antibodies, andtheir use in the treatment and diagnosis of neoplasms. We havecharacterized several human monoclonal antibodies (PM-1, PM-2, and CM-2)that specifically recognize a number of carcinomas. Not only do thesemonoclonal antibodies recognize these neoplasms, but, upon binding to acell, they can induce apoptosis of neoplastic cells, inhibit theirproliferation, or even both. Thus, the PM-1, PM-2, and CM-2 monoclonalantibodies, and other antibodies, or fragments thereof, that arespecific for the antigen recognized by these antibodies, may be used ina variety of methods for diagnosing and treating a neoplasm.

The cell lines that produce the human PM-1, PM-2, and CM-2 monoclonalantibodies were deposited on Jul. 2, 2003 at the German Collection ofMicroorganisms and Cell Cultures (“DSMZ”—Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124Braunschweig, Germany) under the terms of the Budapest Treaty.

Antibodies and Polypeptides

Antibodies play an essential role in maintaining the health of anindividual. In particular, antibodies are present in serum and bind toand help eliminate diverse pathogens such as bacteria, viruses, andtoxins. Antibodies consist of Y-shaped protein structures built from twoheavy chains and two light chains. Each chain has a modularconstruction: each light chain consists of two domains, and each heavychain has at least four domains. The antigen binding site is fashionedby one domain from the heavy chain (V_(H) domain) and one domain fromthe light chain (V_(L) domain). Indeed, small antigen binding fragmentscan be prepared by linking these two domains, either associatednon-covalently, or covalently via disulphide bonds or a peptide linker.The antigen binding domains are more variable in amino acid sequencethan the other domains of the antibody, and are therefore termedvariable (V) domains, in contrast to the constant (C) domains. Theconstant domains of the antibody are responsible for triggering antibodyeffector mechanisms, such as complement lysis and cell-mediated killing.

Antibodies are made by B-lymphocytes in a process involving generearrangement. During the development of these cells, the genes encodingthe variable domains are assembled from genetic elements. In the case ofthe V_(H) domains there are three elements, the un-rearranged V_(H)gene, D segment, and J_(H) segment. In the case of the V_(L) domains,there are two elements, the un-rearranged V_(L) (V Lambda or V Kappa)gene and the J_(L) (J Lambda or J Kappa) segment. Random combination ofthese gene segments and random combination of the rearranged V_(H) andV_(L) domains generate a large repertoire of antibodies, capable ofbinding to a large diversity of equally diverse antigens. Further, theV_(H) and V_(L) regions each have three Complement Determining Regions(CDR) and four framework regions (FR). The FRs are the backbone of theantibody and the CDRs are the parts of the antibody that bind theantigen. One skilled in the art can determine the FR and CDR regions ofan antibody by comparing the amino acid sequence of a number ofantibodies raised in the same species.

In general, the presently claimed polypeptide is any agent that binds toany one of HT-29, CACO-2, COLO-320, COLO-206F, ASPC-1, or BXPC-3, butdoes not bind to non-neoplastic cells. The polypeptide may be anantibody, such as a human monoclonal antibody (e.g., PM-1, PM-2, orCM-2), or a functional fragment thereof. Overall, the polypeptide of theinvention can exclusively bind to both neoplastic tissues and neoplasticcells, but not to non-neoplastic tissue or cells. The polypeptide alsomay induce apoptosis of a neoplastic cell to which it binds, but not ina non-neoplastic cell, or, alternatively, the polypeptide may inhibitproliferation of the neoplastic cell it binds to, but not in anon-neoplastic cell. Desirably, the polypeptide can simultaneouslyinduce apoptosis and inhibit proliferation of neoplastic cells, but notof non-neoplastic cells. Such a polypeptide is, therefore, useful forthe detection, monitoring, prevention, and treatment of cancers inmammals. Exemplary cancers amenable to the methods of the currentinvention include colorectal cancer, ovarian carcinoma, squamous celllung carcinoma, small cell lung carcinoma, lobular and ductal mammarycarcinomas, melanoma, breast cancer, lung cancer, such as lungadenocarcinomas, gastric cancer, pancreatic cancer, such as pancreaticadenocarcinomas, glioma, sarcomas, gastrointestinal cancer, brain tumor,esophageal cancer, such as esophagial squamous cell carcinomas, stomachcancer, osteosarcoma, fibrosarcomas, urinary bladder cancer, prostatecancer, such as prostate adenocarcinomas, renal cancer, ovarian cancer,testicular cancer, endometrial cancer, cervical cancer, uterineadenocarcinomas, Hodgkin's disease, lymphomas, and leukemias. Suchpolypeptides are particularly useful for the detection and treatment ofa stomach adenocarcinoma, colorectal adenocarcinoma, squamous cell lungcarcinoma, lung adenocarcinoma, squamous cell carcinoma of theesophagus, adenocarcinoma of the pancreas, urothel carcinoma of theurinary bladder, renal cell carcinoma of the kidney, adenocarcinoma ofthe prostate, ductal carcinoma of the breast, lobular carcinoma of thebreast, adenocarcinoma of the ovary, adenocarcinoma of the endometrium,or adenocarcinoma of the uterus.

Production

The polypeptides according to the claimed invention can be produced byany method known in the art for small scale, large scale, or commercialproduction of polypeptides. For example, monoclonal antibodies, such asPM-1, PM-2, and CM-2, may be produced by hybridoma cell lines. Such celllines are typically generated by the fusion of spleen and lymph nodelymphocytes derived from patients having a neoplasm, such as coloncarcinoma or a pancreatic carcinoma, with a heteromyeloma cell line.Exemplary heteromyeloma cell lines include, for example, HAB-1 (Vollmerset al, Cancer 74:1525-1532, 1994), CB-F7 (Delvig et al., Hum. AntibodiesHybridomas 6:42-46, 1995), K6H6B5 (Delvig et al., Hum. AntibodiesHybridomas 6:42-46, 1995), H7NS.934 (Delvig et al., Hum. AntibodiesHybridomas 6:42-46, 1995), SHM-D33 (Bron et al., Proc. Natl. Acad. Sci.USA 81:3214-3217, 1984), and B6B11 (Borisova et al., Vopr. Virusol.44:172-174, 1999). The ability to generate human monoclonal antibodiesfrom lymphocytes of cancer patients allows the isolation of antibodiesthat are generated by an immune response in the cancer patient to thetumor.

Typically, portions of the lymph nodes or spleen are surgically removedfrom a patient having cancer, such as colon carcinoma or a pancreaticcarcinoma. Lymphocytes may be prepared as cell suspensions by mechanicalmeans and subsequently fused at, for example, a 1:2 or 1:3 ratio with aheteromyeloma cell line under conditions that result in cell fusion. Forinstance, the heteromyeloma cell line HAB-1, which is generated by thefusion of a human lymphocyte with the mouse myeloma NS-0, may be usedfor this purpose. A proportion of lymphocytes isolated from the cancerpatient may also be maintained in culture. These cells serve as a sourceof human autologous cells useful for the initial antibody screeningdescribed below.

Following the fusion of the lymphocytes derived from the cancer patientwith the heteromyeloma cell line, an antibody producing hybridoma ortrioma is generated. Once constructed, hybridomas are generally stablein growth and antibody production in standard and mass cultures (flasks,miniPerm, fermenters, etc.) for several months. Levels of antibodyproduction typically range between 0.01-0.1 mg/mL in flasks and between0.1-0.5 mg/mL in miniPerm. Cell fusion may be achieved by any methodknown in the art, and includes, for example, the use of 40% polyethyleneglycol. Hybridomas may be cultured in media containing HAT(Hypovanthin-aminopterin-thymidin) and after four weeks, supernatantsmay be screened for antibody production using an ELISA assay. Positiveclones may then be tested in attachment inhibition and binding assaysusing autologous cell lines as prepared above. Positive clones furthermay be tested using immunoperoxidase staining of tumor and normaltissues. Thus, clones may be selected on the basis of their reactivitywith autologous and allogeneic neoplastic cells. The antibody may bepurified from mass cultures with use of cation-exchange chromatographyfollowed by gel filtration as described, for example, by Vollmers et al.(Oncology Reports 5:35-40, 1998). Following the production ofantibodies, additional functional and immunohistochemical tests of theantibodies produced by the trioma may be performed. For example, theantibodies produced by the hybridoma can be tested for their ability toinduce apoptosis, inhibit cellular proliferation, or both, relative tountreated control cells. The antibodies can also be tested for theirability to specifically bind the neoplastic cell lines HT-29, CACO-2,COLO-320, COLO-206F, ASPC-1, or BXCP-3, relative to non-neoplasticcells.

Alternatively, the polypeptide, including an antibody, or a fragmentthereof, may be produced by the expression of the polypeptide orantibody in a host cell such as E. coli or yeast, e.g., S. cerevisiae,or a mammalian cell line. For example, an antibody of the invention maybe identified as follows. A nucleic acid sequence encoding an antibody,or a fragment thereof, may be inserted into filamentous bacteriophage togenerate libraries of approximately 10⁷ or more antibodies. Each phageexpresses an antibody on its surface that is encoded by the nucleic acidit contains. Antibodies of the invention may thus be screened anddetected by functional and histochemical assays as described herein, andsuch genes may be subsequently selected and expressed in E. coli. Thissystem is described, for example, in U.S. Pat. No. 5,876,691.

Antibodies, or functional fragments thereof, may also be generatedusing, for example, direct synthesis using recombinant methods. Thesemethods are standard in the art. For example, a nucleic acid sequencemay be amplified using the polymerase chain reaction (PCR). The PCRtechnique is known in the art and is described, for example in U.S. Pat.No. 4,683,195. Using standard methods, and as described herein, thesequence of a monoclonal antibody expressed by a hybridoma may beobtained and functional fragments of the antibody may be amplified. Forexample, whole RNA may be isolated from a hybridoma expressing atumor-specific monoclonal antibody. cDNA may then be generated from theRNA using reverse transcriptase and the cDNAs which contain thefunctional fragments of the variable regions of the heavy and lightchains may be amplified using PCR. The PCR products may then be purifiedand cloned into expression vectors, e.g., plasmid or viral vectors. Manystandard vectors are available and the selection of the appropriatevector will depend on, for example, the size of the DNA inserted intothe vector and the host cell to be transfected with the vector.

The nucleic acid molecules of the invention may be expressed in avariety of standard vectors and host cells. Any promoter that is activein the host cell may be used to express a nucleic acid molecule.Nonetheless, for expression of an antibody or a fragment of an antibodyin a mammalian cell, use of an immunoglobulin gene promoter isdesirable. Methods of introducing a vector into a host cell are standardin the art and include, electroporation, use of synthetic lipidpolymers, e.g., Lipofectin™, use of calcium chloride, and use of DEAEDextran. Such methods are also described in, for example, Ausubel etal., Current Protocols in Molecular Biology, Wiley Interscience, NewYork, 2001; and Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., 1989.

Isolation of Amino Acid Variants of a Polypeptide

Amino acid sequence variants of a polypeptide, such as an antibody,e.g., a PM-1, PM-2, or CM-2 antibody, can be prepared by introducingappropriate nucleotide changes into the DNA encoding the antibody, or byin vitro synthesis of the desired polypeptide. Such variants include,for example, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence of the PM-1, PM-2, or CM-2 antibody. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., the ability to induceapoptosis of a neoplastic cell, but not a non-neoplastic cell, or theability to inhibit the proliferation of a neoplastic cell, but not anon-neoplastic cell. The amino acid changes also may alterpost-translational processes of an antibody, such as changing the numberor position of glycosylation sites, altering the membrane anchoringcharacteristics, or modifying its susceptibility to proteolyticcleavage.

In designing amino acid sequence variants of a polypeptide, such as anantibody, the location of the mutation site and the nature of themutation will depend on characteristic(s) to be modified. The sites formutation can be modified individually or in series, e.g., bysubstituting first with conservative amino acid choices and then withmore radical selections depending upon the results achieved, or deletingthe target residue.

A useful method for identification of specific residues or regions formutagenesis in a polypeptide is called “alanine scanning mutagenesis”and is described, for example, by Cunningham and Wells (Science244:1081-1085, 1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostdesirably alanine or polyalanine) to affect the interaction of the aminoacids with the surrounding aqueous environment in or outside the cell.The domains demonstrating functional sensitivity to the substitutionsthen are refined by introducing further or other variants at or for thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutationneed not be predetermined. For instance, to optimize the performance ofa mutation at a given site, alanine scanning or random mutagenesis maybe conducted at the target codon or region and the expressed variantsare screened for, e.g., the ability to induce apoptosis of a neoplasticcell and not a non-neoplastic cell, or to inhibit the proliferation of aneoplastic cell and not a non-neoplastic cell.

The sites of greatest interest for substitutional mutagenesis includesites identified as affecting the biological activity of a polypeptide.These sites, especially those falling within a sequence of at leastthree other identically conserved sites, may be substituted in arelatively conservative manner. For instance, ala may be substitutedwith val, leu, or ile; arg may be substituted with lys, gln, or asn; asnmay be substituted with gln, his, lys, or arg; asp may be substitutedwith glu; cys may be substituted with ser; gln may be substituted withasn; glu may be substituted with asp; gly may be substituted with pro;his may be substituted with asn, gln, lys, or arg; ile may besubstituted with leu, val, met, ala, or phe; leu may be substituted withile, val, met, ala, or phe; lys may be substituted with arg, gln, orasn; met may be substituted with leu, phe, or ile; phe may besubstituted with leu, val, ile, or ala; pro may be substituted with gly;ser may be substituted with thr; thr may be substituted with ser; trpmay be substituted with tyr; tyr may be substituted with trp, phe, thr,or ser; and val may be substituted with ile, leu, met, or phe.

Conjugation of the Antibody with a Detectable Agent

If desired, the claimed polypeptide such as an antibody (e.g.,monoclonal antibody, such as PM-1, PM-2, or CM-2), or a fragmentthereof, may be linked to a detectable agent to facilitate thepurification of the polypeptide as well as the diagnosis, monitoring, ortreatment of cancer in a mammal in need thereof. The selection ofsuitable detectable agent will depend on the intended use of thepolypeptide and will be apparent to those of ordinary skill in the art.Detectable agents according to the claimed invention include, forexample, protein purification tags, cytotoxins, enzymes, paramagneticlabels, enzyme substrates, co-factors, enzyme inhibitors, dyes,radionuclides, chemiluminescent labels, fluorescent markers, growthinhibitors, and biotin.

A protein purification tag may be conjugated to the polypeptide of theinvention, to facilitate isolation of the polypeptide. Examples of tagsthat can be used include His-tags, HA-tags, FLAG®-tags, and c-Myc tags.An enzymatic or chemical cleavage site may be engineered between thepolypeptide and the tag moiety so that the tag can be removed followingpurification. Suitable toxins include diphtheria toxin, Pseudomonasexotoxin A, ricin, and cholera toxin. Examples of suitable enzyme labelsinclude malate hydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, alcohol dehydrogenase, alpha-glycerol phosphatedehydrogenase, triose phosphate isomerase, peroxidase, alkalinephosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholinesterase. Examples of suitableradioisotopic labels include ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, and ¹⁴C.Desirably, the radioisotope will emit in the 10-5,000 kev range, moredesirably 100-500 kev. Paramagnetic isotopes may also be conjugated tothe polypeptide and used in vivo for the diagnosis and treatment ofcancer. The use of such conjugated antibodies may be for in vivo nuclearmagnetic resonance imaging. Such a method has previously been described(see, for example, Schaefer et al., JACC 14:472-480, 1989; Shreve etal., Magn. Reson. Med. 3:336-340, 1986; Wolf, Physiol. Chem. Phys. Med.NMR 16:93-95, 1984; Wesbey et al., Physiol. Chem. Phys. Med. NMR16:145-155, 1984; and Runge et al., Invest. Radiol. 19:408-415, 1984).Alternatively, the radiolabeled antibody may also be used inradioimmunoguided surgery (RIGS), which involves the surgical removal ofany tissue the labeled antibody binds to. Thus, the labeled antibodyguides the surgeon towards neoplastic tissue by distinguishing it fromnon-neoplastic tissue. Radiolabels useful for tumor imaging arepreferably short-lived radioisotopes. Various radioactive metals withhalf-lives ranging from 1 hour to 11.4 days are available forconjugation to antibodies, such as scandium-47 (3.4 days), gallium-67(2.8 days), gallium-68 (68 minutes), technetium-99m (6 hours),indium-111 (3.2 days), and radium-223 (11.4 days), of which gallium-67,technetium-99m, and indium-111 are preferable for gamma camera imaging,gallium-68 is preferable for positron emission tomography, andscandium-47 and radium-223 (and other alpha-emitting radionuclides) arepreferable for tumor therapy.

Examples of suitable fluorescent markers include fluorescein,isothiocyalate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,ophthaldehyde, and fluorescamine. Examples of chemiluminescent markersinclude a luminal label, isoluminal label, aromatic acridinium esterlabel, imidazole label, acridinium salt label, oxalate ester label,luciferin label, luciferase label, and aequorin label. Those of ordinaryskill in the art would know of other suitable labels, which may beemployed in accordance with the present invention. Conjugation of thesedetectable agents to the claimed polypeptides such as monoclonalantibodies, or fragments thereof, can be accomplished using standardtechniques commonly known in the art. Typical antibody conjugationtechniques are described by Kennedy et al. (Clin. Chim. Acta 70, 1-31,1976) and Schurs et al. (Clin. Chim. Acta 81, 1-40 , 1977) and include,for example, the glutaraldehyde method, the periodate method, thedimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide estermethod. Antibodies may be radiolabeled by any of several techniquesknown to the art, described, for example, in U.S. Pat. No. 4,444,744.All of these methods are incorporated by reference herein.

In all aspects of the present invention, it is understood that mixturesof different or the same labeled polypeptides specific to differentantigens or different epitopes of the same antigen associated with thesame or different tumor or tumor cell types may be used. Such acombination may enhance detection, localization and/or therapy incertain cases, and can also increase the range of a broad screen formore than one neoplasm or type of neoplasm.

Polypeptides Conjugated to Anti-Tumor Agents

Although the polypeptide of the invention may induce apoptosis ofneoplastic cells, inhibit cellular proliferation of neoplastic cells, orboth, the polypeptide may in addition be conjugated to an agent thatkills neoplastic cells or that inhibits their proliferation. Thetargeting ability of the polypeptide, such as an antibody or fragmentthereof, results in the delivery to deliver of the cytotoxic oranti-proliferative agent to the tumor to enhance the destruction of thetumor. The polypeptide therefore may be used for the treatment andprevention of cancer in a mammal, such as a human patient. The cytotoxicagent linked to the polypeptide may be any agent that destroys ordamages a tumor cell or tumor to which the polypeptide has bound.Examples of such agents include chemotherapeutic agents orradioisotopes, enzymes which activates a pro-drug, or a cytokine.

Suitable chemotherapeutic agents are known to those skilled in the artand include, for example, taxol, mithramycin, deoxyco-formycin,mitomycin-C, L-asparaginase, interferons (especially IFN-alpha),etoposide, teniposide, anthracyclines (e.g., daunomycin anddoxorubicin), methotrexate, vindesine, neocarzinostatin, cis-platinum,chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan, ricin,and calicheamicin. The chemotherapeutic agents may be conjugated to theantibody using conventional methods known in the art.

Suitable radioisotopes for use as cytotoxic agents are also known tothose skilled in the art and include, for example, ¹³¹I, or an astatinesuch as ²¹¹At. These isotopes may be attached to the polypeptide, eithercovalently or non-covalently, using conventional techniques known in theart.

Alternatively, the cytotoxic agent may also be an enzyme, whichactivates a pro-drug. This allows the conversion of an inactive pro-drugto its active, cytotoxic form at the tumor site and is called“antibody-directed enzyme pro-drug therapy” (ADEPT). Thus, thepolypeptide-enzyme conjugate may be administered to the patient andallowed to localize in the region of the tumor to be treated. Thepro-drug is then administered to the patient such that conversion to thecytotoxic drug is localized in the region of the tumor to be treatedunder the influence of the localized enzyme. An exemplary enzyme isbacterial carboxypeptidase G2 (CPG2) the use of which is described in,for example, WO 88/07378. The polypeptide-enzyme conjugate may, ifdesired, be modified in accordance with the teaching of WO 89/00427,such as to accelerate its clearance from areas of the body that are notin the vicinity of a neoplasm. The polypeptide-enzyme conjugate may alsobe used in accordance with WO 89/00427, for example, by providing anadditional component, which inactivates the enzyme in areas of the bodythat are not in the vicinity of the tumor.

As another alternative, the cytotoxic agent conjugated to the claimedpolypeptide may also be a cytokine such as interleukin-2 (IL-2),interleukin-4 (IL-4), or tumor necrosis factor alpha (TNF-alpha). Thepolypeptide targets the cytokine to the tumor so that the cytokinemediates damage to or destruction of the tumor without affecting othertissues. The cytokine may be fused to the polypeptide at the DNA levelusing conventional recombinant DNA techniques.

In addition, any inhibitor of cell proliferation. e.g., genistein,tamoxifen, or cyclophosphamide, may be conjugated with a polypeptide ofthe invention.

Dosage

With respect to the therapeutic methods of the invention, it is notintended that the administration of the claimed polypeptide to a patientbe limited to a particular mode of administration, dosage, or frequencyof dosing; the present invention contemplates all modes ofadministration, including intramuscular, intravenous, intraperitoneal,intravesicular, intraarticular, intralesional, subcutaneous, or anyother route sufficient to provide a dose adequate to decrease the numberof neoplastic cells by inducing apoptosis of neoplastic cells, byinhibiting proliferation of tumor cells, or both. The compound(s) may beadministered to the patient in a single dose or in multiple doses. Whenmultiple doses are administered, the doses may be separated from oneanother by, for example, one day, two days, one week, two weeks, or onemonth. For example, the polypeptide (e.g., a monoclonal antibody, suchas PM-1, PM-2, or CM-2) may be administered once a week for, e.g., 2, 3,4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that,for any particular subject, specific dosage regimes should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions. The precise dose will vary dependent on the polypeptideused, the density, on the tumor surface, of the ligand to which thepolypeptide binds, and the rate of clearance of the polypeptide. Forexample, the dosage of the PM-1, PM-2, or CM-2 antibody can be increasedif the lower dose does not provide sufficient anti-neoplastic activity.Conversely, the dosage of the PM-1, PM-2, or CM-2 antibody can bedecreased if the neoplasm is cleared from the patient.

While the attending physician ultimately will decide the appropriateamount and dosage regimen, a therapeutically effective amount of theclaimed polypeptide, such as a monoclonal antibody or a fragmentthereof, may be, for example, in the range of about 0.1 mg to 50 mg/kgbody weight/day or 0.70 mg to 350 mg/kg body weight/week. Desirably atherapeutically effective amount is in the range of about 0.50 mg to20.0 mg/kg, and more desirably in the range of about 0.50 mg to 15.0mg/kg for example, about 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 8.5, 9.0, 10.0, 11.0, 12.0, 13.0,14.0, or 15.0 mg/kg body weight administered daily, every other day, ortwice a week.

For example, a suitable dose is an amount of the polypeptide that, whenadministered as described above, is capable of inducing apoptosis, andis at least 20% above the basal (i.e., untreated) level. In general, anappropriate dosage and treatment regimen provides the active compound(s)in an amount sufficient to provide therapeutic and/or prophylacticbenefit. Such a response can be monitored by establishing an improvedclinical outcome (e.g., more frequent remissions, complete or partial,or longer disease-free survival) in treated patients as compared tonon-treated patients. According to this invention, the administration ofthe polypeptide can induce neoplastic cell apoptosis by at least 20%,40%, 50%, or 75% above that of an untreated control as measured by anystandard assay known in the art. More desirably, apoptosis is induced by80%, 90%, 95%, or even 100% above that of an untreated control.Alternatively, the administration of the polypeptide can inhibitneoplastic cell proliferation by at least 20%, 40%, 50%, or 75% belowthat of an untreated control as measured by any standard assay known inthe art. More desirably, proliferation is inhibited by 80%, 90%, 95%, oreven 100% below that of an untreated control. Most desirably, thepolypeptide can simultaneously inhibit proliferation and induceapoptosis of neoplastic cells relative to untreated control cells. Suchresponses can be monitored by any standard technique known in the art.In general, for pharmaceutical compositions, the amount of antibodypresent in a dose ranges from about 25 μg to 5 mg per kg of host.Suitable dose sizes will vary with the size of the patient, but willtypically range from about 0.1 mL to about 5 mL.

Formulation of Pharmaceutical Compositions

The claimed polypeptide may be administered by any suitable means thatresults in a concentration having anti-neoplastic properties uponreaching the target region. The polypeptide may be contained in anyappropriate amount in any suitable carrier substance, and is generallypresent in an amount of 1-95% by weight of the total weight of thecomposition. The composition may be provided in a dosage form that issuitable for parenteral (e.g., subcutaneous, intravenous, intramuscular,or intraperitoneal) administration route. The pharmaceuticalcompositions may be formulated according to conventional pharmaceuticalpractice (see, e.g., Remington: The Science and Practice of Pharmacy(20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 andEncyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C.Boylan, 1988-1999, Marcel Dekker, New York).

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. If the neoplastic cells are in direct contact with the blood(e.g., leukemias), or if the tumor is only accessible by the bloodstreamthen the intravenous (I.V.) route may be used. In cases in which tumorsgrow in confined spaces such as the pleural cavity or the peritonealcavity, the polypeptide may be directly administered into the cavityrather than into the blood stream. The formulation and preparation ofsuch compositions are well known to those skilled in the art ofpharmaceutical formulation. Formulations can be found in Remington: TheScience and Practice of Pharmacy, supra.

Diagnosis and Monitoring Cancer Progression

As discussed above, the present invention is directed to a method fordetecting or diagnosing a neoplasm in a mammal, preferably a humanpatient. Typically, any neoplasm in which administration of the claimedpolypeptide causes an induction in apoptosis or a reduction inproliferation are amenable to the methods of this invention.

The claimed polypeptides are particularly useful since they are specificto neoplasms or neoplastic cells, but not normal cells or tissue.Accordingly, this polypeptide can bind to neoplastic cells within thetumor, but not the normal surrounding tissue, thus allowing thedetection, the treatment, or both, of a neoplasm in a mammal. Forinstance, one may use a polypeptide of the invention to determine is abiopsy removed the entire tumor by verifying that no cells bound by thepolypeptide remain in the patient or, by verifying that tumor removedfrom the patient is entirely surrounded by cells that are not bound bythe polypeptide.

It is understood that to improve the sensitivity of detection, multipleneoplastic markers may be assayed within a given sample or individual.Thus, polypeptides such as antibodies or functional fragments specificfor different antigens may be combined within a single assay, or inmultiple assays. Further, multiple primers or probes specific toneoplasms may be used concurrently. The selection of markers may bebased on routine experiments to determine combinations that results inoptimal sensitivity.

In Vitro Detection of a Neoplasm

In general, the diagnosis of a neoplasm in a mammal involves obtaining abiological sample from the mammal (e.g., human patient), contacting suchsample with the polypeptide of the invention (e.g., a monoclonalantibody, such as PM-1, PM-2, or CM-2), detecting in the sample thelevel of reactivity or binding of the polypeptide to neoplastic cellsrelative to a control sample, which corresponds to non-neoplastic cellsderived from healthy tissue from the mammal in which the cancer is beingdiagnosed or from another patient known not to have neoplasm. Thus, themethods of this invention are particularly useful for the detection ofearly stage tumors or metastases, which are otherwise undetectable.Accordingly, in addition to diagnosing a neoplasm in a patient, themethods of this invention may also be used to monitor progression of aneoplasm in a mammal. The polypeptides described herein therefore may beused as markers for the progression of a neoplasm. For this purpose, theassays described below, which are used for the diagnosis of a neoplasm,may be performed over time, and the change in the level of reactivepolypeptide(s) evaluated. For example, the assays may be performed every24-72 hours for a period of 6 months to 1 year, and thereafter performedas needed. In general, a neoplasm is progressing in those patients inwhom the level of bound polypeptide detected increases over time. Incontrast, the neoplasm is not progressing when the level of boundpolypeptide either remains constant or decreases with time.Alternatively, as is noted above, the polypeptide of the invention mayalso be used to determine the presence of tumor cells in the mammalfollowing tumor resection by surgical intervention to determine whetherthe tumor has been completely removed from the mammal.

Desirably, the polypeptide is linked to a detectable agent, whichfacilitates detection, or measurement of polypeptide reactivity. Thebiological sample is any biological material, which may containneoplastic cells and include, for example, blood, saliva, tissue, serum,mucus, sputum, urine, or tears. The biological sample may also be atissue section, which may be fixed tissue, fresh tissue, or frozentissues. A neoplasm is detected or diagnosed in the mammal from whichthe sample was obtained if there is an increase in the level ofreactivity of the antibody with the biological sample over the controlsample. Such increase is at least 10%, 20%, 30%, 40%, 50%, or more than50% over control levels. The level of binding or reactivity can bedetermined by any method known in the art and is described in furtherdetail below.

In Vitro Diagnostic Assays

The diagnosis of neoplasms using the claimed polypeptide may beperformed by any method known to those of ordinary skill in the art forusing a binding agent to detect polypeptide markers in a sample. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the polypeptide may be used forenzyme-linked immunosorbent assay (ELISA), Western blotting or in situdetection of tumor cells in a tissue sample. For example, the ELISAassay typically involves the use of the polypeptide, such as anantibody, immobilized on a solid support to bind to the tumor cells inthe biological sample. The bound tumor cell may then be detected using adetection reagent that contains a reporter group and that specificallybinds to the antibody/tumor cell complex. Such detection reagentsinclude, for example, any binding agent that specifically binds to theantibody, such as an anti-immunoglobulin, protein G, protein A, or alectin. Alternatively, a competitive assay may be utilized, in which thepolypeptide is an antibody and in which the antigens, to which theantibody is specific to is labeled with a reporter group and allowed tobind to the immobilized antibody after incubation of the antibody withthe biological sample. The extent to which components of the sampleinhibit the binding of the labeled antigens to the antibody isindicative of the reactivity of the sample with the immobilizedantibody. Diagnosis of a neoplasm in a patient may also be determined bya two-antibody sandwich assay. This assay may be performed by firstcontacting an antibody that has been immobilized on a solid support,commonly the well of a microtiter plate, with the sample, such thatpolypeptides within the sample are allowed to bind to the immobilizedantibody. Unbound sample is then removed from the immobilizedpolypeptide-antibody complexes and a detection reagent (preferably asecond antibody capable of binding to a different site on thepolypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.For example, to determine the presence or absence of a neoplasm, such ascolorectal adenocarcinoma, the signal detected from the reporter groupthat remains bound to the solid support is generally compared to asignal that corresponds to a predetermined cut-off value. The cut-offvalue for the detection of a neoplasm is the average mean signalobtained when the antibody is incubated with samples from patientswithout a neoplasm.

The method employed for detecting the reporter group depends upon thenature of the reporter group. For radioactive groups, scintillationcounting or autoradiographic methods may be used. Spectroscopic methodsmay be used to detect dyes, luminescent groups and fluorescent groups.Biotin may be detected using avidin, coupled to a different reportergroup (commonly a radioactive or fluorescent group or an enzyme). Enzymereporter groups may generally be detected by the addition of substrate(generally for a defined period of time), followed by spectroscopic orother analysis of the reaction products.

The polypeptides of the invention may also be employed histologicallyfor in situ detection or quantitative determination of tumor cells, forexample, by immunofluorescence or immunoelectron microscopy. In situdetection or determination may be accomplished by removing a tissuespecimen from a patient and allowing a labeled antibody to bind to anytumor cell in the specimen. Using such a procedure not only allows thedetection of neoplastic cells in a sample, but also allows for thedetermination of their spatial distribution. As another example, thebiological sample can be a smear of biological material containingneoplastic cells on a slide, and the detection of neoplastic cells inthe biological material is achieved by examining the smear with amicroscope or by fluocytometry.

In Vivo detection of a Neoplasm

Alternatively, the antibody of the invention may also be used in vivofor detecting and localizing a neoplasm. Such a method may involveinjecting a mammal, desirably a human subject, parenterally with apolypeptide of the invention, such as PM-1, PM-2, or CM-2, which hasbeen labeled with a detectable agent, and is described, for instance, inU.S. Pat. No. 4,444,744. For example, the polypeptide can beradiolabeled with a pharmacologically inert radioisotope andadministered to the patient. The activity of the radioisotope can bedetected in the mammal using a photoscanning device, and an increase inactivity relative to a control reflects the detection and localizationof a neoplasm.

Treatment

In addition to the diagnosis and monitoring of neoplasms in mammals, thepresent invention also features methods for treating neoplasms in amammal, desirably a human patient. The method generally involves theadministration of a biologically effective amount of the polypeptide ofthe invention to the patient. The polypeptide is typically administeredto the mammal by means of injection using any routes of administrationsuch as by intrathecal, subcutaneous, submucosal, or intracavitaryinjection as well as for intravenous or intraarterial injection. Thus,the polypeptide may be injected systemically, for example, by theintravenous injection of the polypeptide such as the PM-1, PM-2, or CM-2antibody into the patient's bloodstream or alternatively, thepolypeptide can be directly injected at the site of the neoplasm or at alocation in proximity to the neoplastic cells.

In general, and as discussed above, binding of the polypeptide of theinvention to neoplastic cells results in an induction in apoptosis, areduction in cellular proliferation, or both relative to the controlsample. Alternatively, the antibodies may also activate the complementpathway, which ultimately causes holes to be punctured into the cellularmembrane, resulting in cell death.

If desired, the polypeptides may also be conjugated to drugs or toxinsas described above. Once attached to the cell surface, the conjugate maybe engulfed into the cell cytoplasm where cell enzymes cleave, and,thus, activate or free the drugs or toxins from the conjugate. Oncereleased, the drugs or toxins damage the cell and irreversibly inducecell death. With respect to radiolabeled antibodies, binding toneoplastic cells and the resulting emission of radiation, at a shortdistance from the cell DNA, produces damage to the latter thus inducingcell death in the next replication round. For example, after a neoplasmhas been detected and localized in a subject, a higher dose of labeledantibody, generally from 25 to 250 mCi for ¹³¹I, and preferably from 50nCi to 150 mCi per dose, based on a 70 kg patient weight, is injected.Injection may be intravenous, intraarterial, intralymphatic,intrathecal, or intracavitary, and may be repeated more than once. Itmay be advantageous for some therapies to administer multiple, divideddoses of radiolabeled polypeptides or polypeptide mixtures, e.g., in therange of 20-120 mCi (70 kg patient), thus providing higher cell-killingdoses to the neoplasm without usually effecting a proportional increasein radiation of normal tissues

Therapy using labeled polypeptides is advantageously used as a primarytherapeutic treatment, but may also be used in combination with otheranti-neoplastic therapies, e.g., radiation and chemotherapy, and as anadjunct to surgery. The administration of such conjugated polypeptidesis particularly useful in the case where small metastases cannot besurgically removed.

Combination of a Polypeptide with Other Anti-Neoplastic Therapies

Chemotherapeutic agents and/or radiation and/or surgical removal of theneoplasm can optionally be combined with any of the methods of thepresent invention. Classes of compounds that can be used as thechemotherapeutic agent include: alkylating agents, antimetabolites,natural products and their derivatives, hormones and steroids (includingsynthetic analogs), and synthetics. Examples of alkylating agents (e.g.,nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas and triazenes) include Uracil mustard, Chlormethine,Cyclophosphamide (Cytoxan®), Ifosfamide, Melphalan, Chlorambucil,Pipobroman, Triethylene-melamine, Triethylenethiophosphoramine,Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, andTemozolomide. Antimetabolites (including folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors)may include, for example, Methotrexate, 5-Fluorouracil, Floxuridine,Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,Pentostatine, and Gemcitabine. Natural products and their derivatives(including vinca alkaloids, antitumor antibiotics, enzymes, lymphokinesand epipodophyllotoxins) may also be used and include, for example,Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin,Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, paclitaxel(paclitaxel is commercially available as Taxol, Mithramycin,Deoxyco-formycin, Mitomycin-C, L-Asparaginase, Interferons (especiallyIFN-alpha), Etoposide, and Teniposide. Hormones and steroids (includingsynthetic analogs) include, for example, 17-alpha-Ethinylestradiol,Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,Dromostanolone propionate, Testolactone, Megestrolacetate, Tamoxifen,Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone,Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, orZoladex. Exemplary synthetics (including inorganic complexes such asplatinum coordination complexes) include Cisplatin, Carboplatin,Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone,Levamisole, and Hexamethylmelamine.

Methods and dosages for the safe and effective administration of most ofthese chemotherapeutic agents are known to those skilled in the art. Inaddition, their administration is described in the standard literature.For example, the administration of many of the chemotherapeutic agentsis described in the “Physicians' Desk Reference” (PDR), e.g., 1996edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA), thedisclosure of which is incorporated herein by reference.

The following examples are provided for the purpose of illustrating theinvention and should not be construed as limiting.

Example 1 Materials and Methods Cell Culture

In this study the following human cell lines were used: BXPC-3(pancreatic adenocarcinoma; ATCC (American Type Culture Collection, P.O.Box 1549, Manassas, Va. 20108) Accession No. CRL-1687), CACO-2 (colonadenocarcinoma), Colo-206F (colon carcinoma). The cell lines werecultured in RPM1-1640 media (PAA, Vienna, Austria) supplemented with 10%fetal calf serum (FCS), 2 mM glutamine and penicillin/streptomycin (both1%) and incubated in a humidified, 5% CO₂ atmosphere at 37° C. For theassays described, cells were grown to sub-confluency, detached withtrypsin/EDTA and washed twice with phosphate-buffered saline (PBS)before use.

Producing Hybridomas

We immortalized lymphocytes by fusing them to the HAB-1 heteromyeloma asfollows.

We washed the HAB-1 heteromyeloma cells twice with RPMI 1640 (PAA,Vienna, Austria) without additives and centrifuged the cells for 5minutes at 1500 rpm. We then thawed frozen lymphocytes obtained fromeither the spleen or the lymph nodes and we washed these cells twicewith RPMI 1640 without additives and centrifuged these cells at 1500 rpmfor 5 minutes. Both the HAB-1 and the lymphocyte cell pellets wereresuspended in 10 ml RPMI 1640 without additives and were counted in aNeubauer cell counting chamber. We washed the cells again, added theHAB-1 cells and the lymphocytes together in a ratio of 1:2 to 1:3, mixedthem, and centrifuged the mixture for 8 minutes at 1500 rpm. Wepre-warmed Polyethylene Glycol 1500 (PEG) to 37° C. and carefully letthe PEG run drop-wise onto the pellet while slightly rotating the 50 mltube. Next, we gently resuspended the pellet and rotated the tube forexactly 90 seconds in a 37° C. water bath. We washed the cells twicewith a full 10 ml pipette of RPMI without additives and centrifuged thecells for 5 minutes at 1500 rpm. We added 1 ml of RPMI 1640 with HATsupplement (PAA, Vienna, Austria) and 10% FCS, 1% glutamine, and 1%penicillin/streptomycin (“RPMI 1640 HAT”) into each well of a 24-wellplate.

The cell pellet was dissolved in RPMI 1640 HAT and 0.5 ml of the cellswas added to each well of the 24-well plate. We then placed the 24-wellplates into a 37° C. incubator and changed the RPMI 1640 HAT mediumweekly. After four to six weeks, the cell culture supernatants werescreened for antibody production in an enzyme-linked immunosorbent assay(ELISA).

Using this protocol, approximately 80% to 90% of the triomas generatedare viable and approximately 50% secrete immunoglobulins. Positiveclones were tested immunohistochemically on autologous tumor tissuesections and clones that showed a positive reaction were subsequentlyre-cloned.

cDNA Synthesis and RT-PCR

To obtain the sequence of the antibody, we isolated whole RNA from thetrioma using the RNASE Kit from Qiagen. Total RNA may also be preparedusing methods standard in the art, e.g., those described in Krenn et al.(Clin. Exp. Immunol. 115:168-175, 1999). cDNA synthesis from total RNAobtained from hybridoma cell lines PM-1, PM-2, and CM-2 was performedwith 5 μg total RNA using Gibco BRL (Eggenstein, Germany) M-MLV ReverseTranscriptase according to the manufacturer's instructions. Theamplification of V_(H) and V_(L) genes was carried out in a 25 μl volumewith 1.75 mM MgCl₂, 0.4 pM primer, 200 μM of each dNTP, and 1U Taqpolymerase (MBI Fermentas, St. Leon-Rot, Germany). The PCR-products wereamplified using the following cycle profiles: 95° C. for 2 min, followedby 35 cycles of 94° C. for 30 sec; 65° C. for 30 sec (for VH3 and VH4primers), 60° C. for VH1, VH2, VH5, VH6 and 52° C. for VL primersrespectively; a final extension at 72° C. for 4 min.

Sequencing the Antibody

The PCR products were purified using gel electrophoresis through 2%agarose (Roth, Karlsruhe, Germany) followed by gel extraction of the PCRproduct using a Jetsorb gel extraction kit (Genomed, Bad Oeynhausen,Germany). The PCR products were then cloned using the pCR-Script Amp SK⁺cloning kit (Stratagene, Heidelberg, Germany). Ten positive clones weresequenced using the DyeDeoxy termination cycle sequencing kit (AppliedBioSystems Inc., Weiterstadt, Germany) and analysed with an ABIPrism373automated DNA sequencer (both strands were sequenced using T3 and T7primers). The sequences were analysed using the DNASIS for Windowssequence comparison software and the GenBank and IMGT/V-QUEST databases.The International Immunogenetics (“IMGT”) database is coordinated byMarie-Paule Lefranc at the Université Montpellier, Montpellier, France.

Immunohistochemical Staining of Paraffin Sections

Paraffin-embedded human tissues were sectioned (2 μm), the paraffin wasremoved as follows:

Two xylene washes for 5 minutes each,

Two 100% ethanol washes for 5 minutes each,

Two 90% ethanol washes for 5 minutes each,

Two 70% ethanol washes for 5 minutes each, and

Three washes in distilled H₂O.

The slides containing the tissue sections were incubated in 75 mldistilled H₂O and 25 ml de-masking solution (Demaskierungslösung G,Biologo, Kronshagen, Germany) in a preheated water-bath at 100° C. for20 minutes. The slides were placed into Tris/NaCl (3 grams Tris, 40.5grams NaCl in 5 litres of distilled H₂O and pH adjusted to 7.4 with HCl)for 5 minutes, blocked for 15-30 minutes with 150 μl of 0.5% BovineSerum Albumin Fraction V (“BSA;” Roth, Karlsruhe, Germany) in phosphatebuffered saline (“PBS”) per slide, and washed once with Tris/NaCl.

The sections were incubated with PM-1, PM-2, and CM-2 antibodies, andunrelated, human monoclonal IgM antibodies (ChromPure IgM, Dianova,Hamburg, Germany, 10 μg/ml) or mouse CAM 5.2 antibody diluted 1:50 withBSA/PBS (Dako, Hamburg, Germany) for 2.5 hours in a humidified incubatorat 37° C. The sections were then washed three times with Tris/NaCl (3grams Tris, 40.5 grams NaCl in 5 litres of distilled H₂O and pH adjustedto 7.4 with HCl), followed by incubation with peroxidase-labeled rabbitanti-human or rabbit anti-mouse conjugate (Dako) diluted 1:50 in PBScontaining 30% rabbit serum (for antibody 103/51) at RT for 1 hour.After washing three times with Tris/NaCl the tissue sections wereincubated in PBS for 10 minutes before staining with diaminobenzidine(0.05%)-hydrogen peroxide (0.02%) for 10 minutes at room temperature(RT). The reaction was stopped using running tap water and the sectionscounterstained with hematoxylin. After mounting with glycerol-gelatin,the sections were analyzed using light microscopy.

Immunohistochemical Staining of Cryo-Sections from Autologous Tumors

Frozen human tissues were sectioned (4 μm), fixed in acetone, air-driedand washed with Tris/NaCl (3 grams Tris, 40.5 grams NaCl in 5 litres ofdistilled H₂O and pH adjusted to 7.4 with HCl). The cryo-sections werethen blocked with PBS containing 3% milk powder for 30 minutes at RT.After washing three times with Tris/NaCl the sections were incubatedwith PM-1, PM-2, or CM-2 human IgM antibodies, unrelated humanmonoclonal IgM (Chrompure IgM, Dianova, 10 μg/ml) or mouse CAM 5.2antibody diluted 1:50 with BSA/PBS (Dako) for 30 minutes at RT. Thesections were washed three times with Tris/NaCl, followed by incubationwith secondary antibodies (peroxidase-labeled rabbit anti-human orrabbit anti-mouse conjugate 1:50) for 30 minutes at RT. After washingthree times with Tris/NaCl and incubation in PBS for 10 minutes, thesections were stained with diaminobenzidine (0.05%)-hydrogen peroxide(0.02%) for 10 minutes at RT. The reaction was stopped under running tapwater and the sections counterstained with hematoxylin. After mountingwith glycerol-gelatin, the sections were analyzed using lightmicroscopy.

Preparation of Tumor Cell Membrane Extracts

Isolation of membrane proteins from tumor cells was performed asdescribed using standard methods in the art, as described, for example,in Hensel et al. (Int. J. Cancer 81:229-235, 1999). In particular,confluent tumor cells (BXPC-3 for PM-1 and PM-2, CACO-2 for CM-2) werewashed twice with PBS, harvested with a cell scraper, centrifuged, andresuspended in hypotonic buffer (20 mM HEPES, 3 mM KCl, 3 mM MgCl₂) andincubated for 15 minutes on ice. The cells were then sonicated for 5minutes and the nuclei were pelleted by centrifugation at 10,000×g for10 min. The supernatant was centrifuged for 40 minutes at 100,000×g in aswing-out rotor to pellet the membranes. After washing the pellet withhypotonic buffer, the pellet was resuspended in membrane lysis buffer(50 mM HEPES pH 7.4, 0.1 mM EDTA, 10% glycerol, and 1% Triton X-100).Complete protease inhibitor (Boehringer, Mannheim, Germany) also wasadded to all solutions.

Western Blotting

Western blots were preformed using standard techniques as described, forexample, in Hensel et al. (Int. J. Cancer 81:229-235, 1999). In short,blotted nitrocellulose membranes were blocked with PBS containing 3% lowfat milk powder, followed by incubation for 1 hour with 20-40 μg ofPM-1, PM-2, or CM-2 human IgM antibodies or unrelated human control IgM(ChromPure IgM, Dianova). The secondary antibody (peroxidase-coupledrabbit anti-human IgM antibody 1:1,000, Dianova) was detected with theSUPERSIGNAL chemiluminescence kit from Pierce (KMF, St. Augustin,Germany).

Cytospin Preparation

The adherent growing cells were detached by adding Trypsin/EDTA (PAA,Vienna, Austria) followed by a 5 minute incubation in an humidifiedincubator (37° C., 5% CO₂) and centrifugation for 5 minutes at 1,500rpm. The cells then were washed twice with 10 ml of RPMI-1640 cellculture medium (PAA, Vienna, Austria). The cell number was adjusted to adensity of 1×10⁵ cells/ml. From this solution, 100 μl were centrifugedonto microscope slides with a cytospin centrifuge (CYTOSPIN 2, Shandon,UK) for 2 minutes at 50 rpm. The resultant cytospins were dried for atleast 2 hours and stained as specified below.

Immunoperoxidase Staining of Cytospins and Cryosections

Cytospins were dried for at least two hours at room temperature orcryosections were dried for at least two hours after they were cut. Thesections or cytospins were then fixed for 10 minutes in acetone. Thefixed cryosections/cytospins were dried for 30 minutes at roomtemperature, washed three times with Tris-NaCl (3 grams Tris, 40.5 gramsNaCl in 5 litres of distilled H₂O and pH adjusted to 7.4 with HCl), andplaced into Tris/NaCl for 5 minutes. The cryosections/cytospins wereblocked for 15-30 minutes with 3% milk powder in PBS (100 μl percryosection/cytospin) and washed three times with Tris-NaCl. Thecryosections/cytospins were incubated in 100 μl of primary antibody percryosection/cytospin (e.g., at 20 μg/ml in 0.5% BSA/PBS; CK 8 at 1:50 inBSA/PBS; CAM 5.2 at 1:10 in BSA/PBS; or RPMI 1640 media (PAA, Vienna,Austria) as a negative control) for 30 minutes in a humidified chamberat room temperature. Following the incubation, thecryosections/cytospins were washed three times with Tris-NaCl.

The cryosections/cytospins were then incubated in 100 μl of a solutioncontaining the secondary antibody (70% PBS+30% rabbit or human serum+e.g., 1:50 rabbit anti-mouse antibody, peroxidase coupled or 1:50 rabbitanti-human IgM antibody, peroxidase coupled; Dako, Hamburg, Germany) percryosection/cytospin for 30 minutes in a humidified chamber at roomtemperature and washed three times with Tris-NaCl and placed into PBSfor 10 minutes. The cryosections/cytospins where then incubated for 10minutes in 100 μl of a solution containing 0.05% diaminobenzidine and0.02% hydrogen peroxide (Sigma, Taufkirchen (München), Germany).Following the incubation, the cryosections/cytospins were washed withdistilled H₂O and placed into a hematoxylin staining solution (Roth,Karlsruhe, Germany) for 5 minutes. The cryosections/cytospins were thenrinsed for 15 minutes under running tap water, washed with distilledH₂O, and cover with pre-warmed glycerol-gelatin.

The following experiments were carried out using the above materials andmethods.

Example 2 Generation of the Cell Line Expressing the PM-1, PM-2, or CM-2Monoclonal Antibody

As described above, we obtained the PM-1, PM-2, or CM-2 monoclonalantibody expressing hybridoma by fusing lymphocytes obtained from thespleen or lymph nodes of a cancer patient with the heteromyeloma cellline HAB-1 (Faller, et al., Br. J. Cancer 62:595-598, 1990). Thelymphoid sources were not pre-selected in terms of the age or sex of thepatient. The resultant cell is a type of hybridoma known as a trioma, asit is the fusion of three cells. Like normal B-lymphocytes, this triomahas to ability to produce antibodies. The specificity of the antibody isdetermined by the specificity of the original lymphocyte from thepatient that was used to generate the trioma.

The hybridoma supernatants were screened for antibody production usingan ELISA assay. Following ELISA, antibodies were primarily testedimmunohistochemically against their autologous tumor for tumor specificreactivity. Antibodies PM-1 and PM-2 were generated from the lymphocytesof a pancreatic cancer patient and the CM-2 antibody was generated fromthe lymphocytes of a patient with colon carcinoma.

The amino acid sequence (SEQ ID NO:1) and the nucleic acid sequence (SEQID NO:2) of the variable region of the light chain of human monoclonalantibody PM-1 are shown in FIG. 12. As indicated in FIG. 12, ComplementDetermining Region 1 (CDR1) of the PM-1 variable region light chainspans nucleotides 76-93 which encode amino acids 26-31, CDR2 spansnucleotides 145-153 which encode amino acids 49-51, and CDR3 spansnucleotides 262-285, which encode amino acids 88-95.

The amino acid sequence (SEQ ID NO:3) and the nucleic acid sequence (SEQID NO:4) of the variable region of the heavy chain of human monoclonalantibody PM-1 are shown in FIG. 13. As indicated in FIG. 13, CDR1 of thePM-1 variable region heavy chain spans nucleotides 31-54 which encodeamino acids 11-18, CDR2 spans nucleotides 106-129 which encode aminoacids 36-43, and CDR3 spans nucleotides 244-270 which encode amino acids82-90.

The amino acid sequence (SEQ ID NO:5) and the nucleic acid sequence (SEQID NO:6) of the variable region of the light chain of human monoclonalantibody PM-2 are shown in FIG. 14. As indicated in FIG. 14, CDR1 of thePM-2 variable region light chain spans nucleotides 76-102 which encodeamino acids 26-34, CDR2 spans nucleotides 154-174 which encode aminoacids 52-58, and CDR3 spans nucleotides 289-309, which encode aminoacids 97-103.

The amino acid sequence (SEQ ID NO:7) and the nucleic acid sequence (SEQID NO:8) of the variable region of the heavy chain of human monoclonalantibody PM-2 are shown in FIG. 15. As indicated in FIG. 15, CDR1 of thePM-2 variable region heavy chain spans nucleotides 31-54 which encodeamino acids 11-18, CDR2 spans nucleotides 106-129 which encode aminoacids 36-43, and CDR3 spans nucleotides 244-300, which encode aminoacids 82-100.

The amino acid sequence (SEQ ID NO:9) and the nucleic acid sequence (SEQID NO:10) of the variable region of the light chain of human monoclonalantibody CM-2 are shown in FIG. 16. As indicated in FIG. 16, CDR1 of theCM-2 variable region light chain spans nucleotides 76-102 which encodeamino acids 26-34, CDR2 spans nucleotides 151-162 which encode aminoacids 51-54, and CDR3 spans nucleotides 271-297, which encode aminoacids 91-99.

The amino acid sequence (SEQ ID NO:11) and the nucleic acid sequence(SEQ ID NO:12) of the variable region of the heavy chain of humanmonoclonal antibody CM-2 are shown in FIG. 17. As indicated in FIG. 17,CDR1 of the CM-2 variable region heavy chain spans nucleotides 46-66which encode amino acids 16-22, CDR2 spans nucleotides 118-141 whichencode amino acids 40-47, and CDR3 spans nucleotides 256-300, whichencode amino acids 86-100.

Example 3 Immunohistochemical Characterization of an Antibody

To characterize the monoclonal antibody secreted by a hybridoma, wetested the antibody against a panel of normal and tumor tissues using animmunoperoxidase assay as described in the materials and methods. Thisassay provided us with an overview of which tissues were stained by theantibody and of the distribution of the antigen.

Antibodies that are specific for tumor cells and not for normal tissuewere further characterized. First, we tested these antibodies againstthe same types of tumors from different patients. We then tested theseantibodies against tumors of other organs and, finally, against normaltissues. Using these assays, we identified the human PM-1, PM-2, andCM-2 monoclonal antibodies. The tumor reactive antibodies generated anddescribed in this study are of the IgM/λ isotype (see Table 1).

TABLE 1 Origin of Monoclonal IgM Antibodies and Clinical Data of CancerPatients Tumour Tumour Source of Ig Antibody Organ Tumour type stagegrade Age Sex Lymphocytes Class PM-1 Pancreas Adenocarcinoma T1N1 G2-347 M Spleen IgM/λ PM-2 IgM/λ CM-2 Colon Adenocarcinoma T2N0 G2 78 MSpleen IgM/λ

To investigate the genetic origin of these human monoclonal IgMantibodies the V_(H) and V_(L) genes were amplified, cloned andsequenced. The sequences were compared with germ-line sequences in theIMGT/V-QUEST database to identify the most homologous germ-line genesand to detect somatic mutations. The results are represented in Table 2.The degree of identity of the nucleotide sequences of the V_(H) segmentto those of the closest reported germ-line V_(H) genes ranged from 97.2to 100% as summarized in Table 2.

TABLE 2 Characterization of Variable Heavy and Light Chain Regions ofMonoclonal IgM Antibodies Heavy chain Light chain Germ-line Homology R/SR/S Germ-line Homology R/S R/S Antibody gene (%) Frame CDR gene (%)Frame CDR PM-1 IGHV3- 100 0/0 0/0 IGLV3- 99.0 2/1 0/0 23*01 10*01 PM-2IGHV3- 100 0/0 0/0 IGLV5- 98.2 3/2 0/0 23*01 45*01 CM-2 IGHV5- 97.2 2/13/2 IGLV2- 97.2 0/2 4/2 51*01 14*01

Different VH genes of the VH3, VH4 and VH5 gene family expressed theantibodies. The high homology of the VH regions to the germ-line genesand the low R/S ratio, which is an indicator for affinity maturation ofantibodies, indicates that none of the antibodies underwent affinitymaturation by somatic mutation due to antigen contact. The degree ofidentity of the nucleotide sequences of the V_(L) segment to their mosthomologous V_(L) germ-line genes ranged from 97.2 to 99.0%, with allfive antibodies utilizing λ-light chain genes. The R/S ratio is againlow and with one exception (antibody CM-2) restricted to the frameworkregion. The data indicate that all three antibodies belong to the familyof naturally occurring, non-affinity matured antibodies.

After initial testing on autologous tumors, the reaction patterns of theantibodies were investigated in greater detail using immunohistochemicalstaining on a variety of paraffin- and cryo-embedded carcinomas andnormal tissues. The PM-1, PM-2, and CM-2 antibodies exhibited no bindingactivity with normal tissues (Table 3).

TABLE 3 Reaction Pattern of the Monoclonal IgM Antibodies on NormalTissues M6 CAM (IgM- Tissue PM-1 PM-2 CM-2 5.2 Control) Stomach − − − +− Colon − − − + − Lung − − − − − Esophagus − − − − − Urinary bladder − −− − − Prostate − − − − − Breast − − − − − Pancreas − − − − − SmallIntestine − − − + −

In addition, the CM-2 antibody failed to stain the following normaltissues: thyroid, aorta, myocardium, pericardium, tongue, smallintestine, corpus pineal, pituitary, bone marrow, blood, cerebellum,rectum, thymus, tonsilla palatina, lymph nodes, adrenal gland, ductusdeferens, ovary, tuba uterina, corpus uteri, cervix uteri (portio vag.),skin, skeletal muscle, placenta, spinal marrow, and cerebral cortex. TheCM-2 antibody also failed to stain the following fetal tissues: lung,stomach, ileum, pancreas, liver, spleen, thymus, kidney, spinal marrow,cerebral cortex, cerebellum, corpus pineal, and pituitary.

In contrast, the antibodies show a very heterogeneous reactivity patternwith tumor tissues. The PM-2 antibody, for example, reacts with a largenumber of the carcinomas tested in this study, whereas the reactivity ofantibody CM-2 is more restricted (for details see Table 4).

TABLE 4 Reaction Pattern of the Monoclonal IgM Antibodies on TumorTissues Carcinoma PM-1 PM-2 CM-2 M6 (IgM- Tissue type +/− +/− +/− CAM5.2Control) Stomach Adeno 3/1 3/0 0/3 + − Colon Adeno 2/1 3/0 11/16 + −Small Intestine 1/0 1/0 n.d. + − Lung Adeno 3/1 3/0 0/4 + − Squamous 2/23/0 0/3 +(CK5/6) − cell Liver 1/1 2/0 0/2 + − Esophagus Squamous 3/0 3/00/3 +(CK5/6) − cell Pancreas Adeno 5/1 6/0 0/6 + − Urinary bladderUrothel 0/1 1/0 0/1 + − Kidney Renal cell 0/1 1/0 0/1 − − Adeno n.d.n.d. 0/2 − Prostate Adeno 1/4 4/1 0/5 + − Breast Invasive 2/1 3/0 0/3 +− (ductal) Invasive 2/1 3/0 0/3 + − (lobular) Ovary Adeno 1/1 3/0 0/2 +− Uterus Adeno 1/2 3/0 0/3 + − Adrenal Gland Adeno 1/0 1/0 n.d. + −

The positive reaction of antibody PM-1 was not restricted toadenocarcinoma of the pancreas as clear positive reactions wereobserved, among others, on squamous cell carcinoma of the lung (FIGS. 1Aand 1B). Antibody PM-2 gave a broad staining pattern on a variety oftumor tissues that were tested (FIGS. 1C and 1D). FIG. 2 shows thereactivity of the CM-2 antibody on a colon carcinoma. As shown in thisFigure, the CM-2 antibody stains cancerous colon tissue. The CM-2antibody also stains adenocarcinomas of the endometrium. In addition tothe tissues shown in Table 4, the CM-2 antibody also fails to stain thefollowing tumor tissues: carcinomas of the thryoid, bronchi (squamouscell, small cell, and large cell), hepatocytes, and cholangio cells, aswell as nephroblastomas, seminomas, yolk sac tumors, teratomas,teratocarcinomas, melanomas, thymomas, fibrosarcomas, myxofibrosarcomas,rhabdomyoscarcomas, leiomyosacromas, neuroblastomas, squamous cellcarcinomas of the oropharynx, and acute myeloid leukemias. The positivecontrol antibody used in these experiments was a mouse monoclonalantibody against human cytokeratin 5/6 (“CK 5/6;” Dako A/S, Denmark) ora mouse monoclonal antibody against human cytokeratin (“CAM 5.2;” BectonDickinson, New Jersey).

To examine the antigens recognized by the antibodies, Western blots wereperformed with membrane extracts of established carcinoma cell lines.The antibodies PM-1 and PM-2 produced two specific bands each onpancreas adenocarcinoma cell line BXPC-3. Antibody PM-1 reacted withmembrane proteins of about 35 and 65 kDa (FIG. 5A), whereas antibodyPM-2 reacted with proteins of about 55 and 115 kDa (FIG. 5B). AntibodyCM-2 reacted with antigens between 40 and 50 kDa (FIG. 5C). To rule outnon-specific binding of IgM antibodies to membrane extracts, unrelatedhuman control IgM was used as control.

Moreover, the PM-1, PM-2, and CM-2 monoclonal antibodies alsospecifically stain a number of carcinoma cell lines. In particular, thePM-1 antibody binds to the ASPC-1 pancreatic carcinoma cell line(American Type Culture Collection (“ATCC”) Accession No. CRL-1682) andthe BXPC-3 pancreatic carcinoma cell line (ATCC Accession No. CRL-1687).The PM-2 antibody specifically binds to the CACO-2 human colorectaladenocarcinoma cell line (ATCC Accession No. HBT-37, DSMZ Accession No.ACC 169), the human colon carcinoma cell line COLO-320 (DSMZ AccessionNo. ACC 144), the human colon carcinoma cell line COLO-206F (DSMZAccession No. ACC 21), the HT-29 human colorectal adenocarcinoma cellline (ATCC Accession No. HTB-38), ASPC-1 pancreatic carcinoma cells, andBXPC-3 pancreatic carcinoma cell line. Further, the CM-2 antibodyspecifically binds to CACO-2 and COLO 206F cells. Slides of these cellswere stained according to the cytospin protocol described in thematerials and methods section.

Example 4 Determining Whether an Antibody Induces Apoptosis

A number of assays standard in the art may be used to determine if anantibody induces apoptosis of a cell.

For example, we used the CELL DEATH DETECTION ELISA^(PLUS) (Roche,Mannheim, Germany) to analyze the extent to which the PM-1, PM-2, andCM-2 antibodies induce apoptosis. The cell death detection ELISA isbased on a quantitative sandwich-enzyme-immunoassay principle usingmouse monoclonal antibodies directed against DNA and histones,respectively. This assay allows the specific determination of mono- andoligo-nucleosomes which are released into the cytoplasm of cells whichdie from apoptosis.

In particular, 1×10⁴ tumor cells (BXPC-3 for PM-1 and PM-2, CACO-2 forCM-2) were plated on 96-well plates and incubated in presence ofdifferent concentrations of the human IgM-antibodies for 24 hours at 37°C. and 7% CO₂ in an CO₂ incubator. Depleted cell culture supernatantwith unrelated IgM antibodies served as negative control. After theincubation period, the cells were centrifuged for 10 minutes and thesupernatants were removed. The resulting cell pellets were thenincubated with lysis-buffer for 30 minutes at room temperature. Aftercentrifugation the supernatants were transferred into astreptavidin-coated microtiter plate (MTP) and immunoreagent (a mixtureof 10% Anti-Histone-Biotin, 10% Anti-DNA-peroxidase (Anti-DNA POD) and80% incubation buffer) added before incubation for 2 hours at roomtemperature on a MTP shaker at 250 rpm. Following the incubation period,unbound components were removed by a washing step with incubationbuffer. POD was determined photometrically with ABTS™ as a substrate (1ABTS™ (2,2′-Azino-di[3-ethyl-benz-thiazolin-sufonat) tablet in 5 mlsubstrate buffer). Antibody-induced apoptosis was measured bydetermining the color intensity of the green precipitate that it formedas a result of this reaction using an ELISA reader at a wavelength of415 nm in comparison to ABTS™ solution as a blank (reference wavelengthof approximately 490 nm). Based on this color intensity, we calculatedthe level of the antibody-induced apoptosis. These experiments clearlyshowed that each antibody, PM-1, PM-2, and CM-2, induces apoptosis incarcinoma cells after 24 hours of incubation (FIGS. 4A and 4B).

In addition, as is shown in FIGS. 7 and 9, the PM-1 and PM-2 monoclonalantibodies, respectively, induce apoptosis in BXCP-3 human pancreaticcarcinoma cells after a 24 hour incubation period when compared to anegative control. The Y-axis in these figures is the difference betweenthe absorbance at 415 nm and at the 490 nm reference wavelength(A₄₁₅-A₄₉₀) and the negative control is RPMI 1460 medium. Theconcentration of the PM-1 antibody was either 16 μg or 32 μg/ml insupernatant and the concentration of the PM-2 antibody was either 6 μgor 12 μg/ml in supernatant.

As is shown in FIG. 11, CM-2 induces apoptosis of CACO-2 humancolorectal carcinoma cells after a 24 hour incubation. Again, the Y-axisin this figure is the difference between the absorbance at 415 nm and atthe 490 nm reference wavelength (A₄₁₅-A₄₉₀). The negative control isRPMI 1460 medium. As is shown in FIG. 11, both a commercially availableCD95 Fas antibody at 2 μg/ml and the supernatant containing the CM-2monoclonal antibody (22 μg/ml) induce apoptosis when compared to thenegative control.

Example 5 Determining Whether an Antibody Inhibits Cell Proliferation

Cell proliferation may be assayed by a number of methods that arestandard in the art, for example, by the reduction of tetrazolium salts.The yellow tetrazolium salt3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (“MTT”)(Sigma, St. Louis, Mo.), is reduced by metabolically active cells, inpart by the action of mitochondrial dehydogenase enzymes to generatereducing equivalents such as NADH and NADPH. The resulting intracellularpurple formazan can be solubilized and quantified by spectrophotometricmeans. The MTT cell proliferation assay measures the rate of cellproliferation and, when metabolic events lead to apoptosis, thereduction in cell viability.

For the MTT assay, we trypsinized cells (BXPC-3 for PM-1 and PM-2,Colo-206F for CM-2) and resuspended the cells in 10 ml of RPMI-1460medium contains 10% Fetal Calf Serum (FCS), 1% glutamine, and 1%penicillin/streptomycin (complete medium). The cells were then countedand diluted to 1×10⁶ cells/ml. 50 μl of this suspension were pipettedinto wells of a 96-well plate, resulting in approximately 5×10⁴cells/well. The first row of wells was left empty. We then added 50 μlof the antibody diluted in complete medium to each well. The 96-wellplate was then incubated for 24 or 48 hours in a 37° C. incubator. Afterthe incubation period, 50 μl MTT solution (5 mg/ml in PBS) were added toeach well. The 96-well plate was incubated for 30 minutes at 37° C. andcentrifuged for 5 minutes at 800×g. The supernatant was aspirated, 150μl of dimethylsulphoxide (DMSO) were added to each well, and the cellpellet was resuspended. Absorption was determined at a wavelength of 540nm and at a reference wavelength of 690 nm in an ELISA reader.

After 24 or 48 hours, the PM-1, PM-2, and CM-2 antibodies inhibited cellproliferation of the respective tumor cell lines in aconcentration-dependent manner, while the controls with depleted cellculture supernatant remained unchanged (FIGS. 3A and 3B).

Further exemplary results of such experiments are depicted in FIGS. 6A,6B, 8A, 8B, 10A, and 10B. FIGS. 6A and 6B show the results ofexperiments using BXPC-3 pancreatic carcinoma cells that were incubatedwith the PM-1 monoclonal antibody, with depleted supernatant, or withoutan antibody for 24 hours (FIG. 6A) or 48 hours (FIG. 6B). The y-axisshows the difference in absorbance at 540 nm and 690 nm (A₅₄₀-A₆₉₀). Asis evident from these graphs, incubation with the PM-1 monoclonalantibody resulted in a decrease in cell proliferation and cell viabilityafter both a 24 hour and a 48 hour incubation period. Similarly, as isshown in FIGS. 8A and 8B, incubation of BXPC-3 cells with the PM-2monoclonal antibody resulted in a decrease in proliferation and cellviability after both a 24 hour (FIG. 8A) and a 48 hour period (FIG. 8B).In addition, as is shown in FIGS. 10A and 10B, incubation of COLO-206Fcolon carcinoma cells with the CM-2 monoclonal antibody resulted in adecrease in proliferation and cell viability after both a 24 hour (FIG.10A) and a 48 hour period (FIG. 10B).

Example 6 In Vivo Imaging of a Neoplasm

A patient suspected of having a neoplasm, such as a colorectalcarcinoma, may be given a dose of radioiodinated PM-1, PM-2, or CM-2antibody, or another tumor-specific polypeptide, and radiolabeledunspecific antibody using the methods described herein. Localization ofthe tumor for imaging may be effected according to the procedure ofGoldenberg et al. (N. Engl. J. Med., 298:1384, 1978). By I.V. aninfusion of equal volumes of solutions of ¹³¹I-PM-1, PM-2, or CM-2antibody and Tc-99m-labeled unspecific antibody may be administered to apatient. Prior to administration of the reagents I.V., the patient istypically pre-tested for hypersensitivity to the antibody preparation(unlabeled) or to antibody of the same species as the antibodypreparation. To block thyroid uptake of ¹³¹I, Lugol's solution isadministered orally, beginning one or more days before injection of theradioiodinated antibody, at a dose of 5 drops twice or three-timesdaily. Images of various body regions and views may be taken at 4, 8,and 24 hours after injection of the labeled preparations. If present,the neoplasm, e.g., a colorectal carcinoma, is detected by gamma cameraimaging with subtraction of the Tc-99m counts from those of ¹³¹I, asdescribed for “^(I)I-labeled anti-CEA antibody and Tc-99m-labeled humanserum albumin by DeLand et al. (Cancer Res. 40:3046, 1980). At 8 hoursafter injection, imaging is usually clear and improves with time up tothe 24 hour scans.

Example 7 Treatment of a Neoplasm Using Labeled Antibody Mixtures

A patient diagnosed with a neoplasm, for example, a female patientdiagnosed with a breast carcinoma, may be treated with the polypeptidesof the invention as follows. Lugol's solution may be administered, e.g.,7 drops 3 times daily, to the patient. Subsequently, a therapeutic doseof ¹³¹I-PM-1, PM-2, or CM-2 antibody may be administered to the patient.For example, a ¹³¹I dose of 50 mCi may be given weekly for 3 weeks, andthen repeated at intervals adjusted on an individual basis, e.g., everythree months, until hematological toxicity interrupts the therapy. Theexact treatment regimen is generally determined by the attendingphysician or person supervising the treatment. The radioiodinatedantibodies may be administered as slow I.V. infusions in 50 ml ofsterile physiological saline. After the third injection dose, areduction in the size of the primary tumor and metastases may be noted,particularly after the second therapy cycle, or 10 weeks after onset oftherapy.

Example 8 Treatment Using Conjugated Antibodies

A patient diagnosed with a neoplasm, for example, a female patient withbreast cancer that has metastasized to the chest and lungs, may betreated with solutions of ¹³¹I-PM-1, PM-2, or CM-2, ¹⁰B-PM-1, PM-2, orCM-2, and a Tc-99m labeled unspecific antibody. An amount of¹³¹I-labeled PM-1, PM-2, or CM-2 antibody (in 50 ml of sterilephysiological saline) sufficient to provide 100 mCi of ¹³¹I activitybased on a 70 kg patient weight may be administered to the patient. Thisdosage is equal to 3.3 mg of an antibody having 40-80 Boron atoms and8-16 Boron-10 atoms per antibody molecule. The neoplasm is firstprecisely localized using the procedure of Example 6. In addition,Lugol's solution should be continuously administered to the patient, asin the previous example. A well-collimated beam of thermal neutrons maythen be focused on the defined tumor locations. Irradiation with anexternal neutron beam dose of 400-800 rads, delivered in a period offrom 8-20 min, is effected for each tumor locus, and is optionallyrepeated with administration of the tumor-locating antibody, with orwithout the radiolabel, at intervals adjusted on an individual basis,but usually not exceeding a total dose of 3200 rads unless simultaneousexternal irradiation therapy is indicated. If desired, in addition tothis therapy, an anti-tumor agent, such as a chemotherapeutic agent, mayalso be administered to the patient.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

German Patent Application Nos. 102 29 907.2, 102 30 516.1, 102 29 906.4,U.S. Pat. Nos. 5,367,060 and 5,641,869, and all other references citedherein are hereby incorporated by reference.

1. A purified polypeptide that induces apoptosis of a neoplastic cell towhich it binds, but does not induce apoptosis of a non-neoplastic cell,wherein said polypeptide specifically binds to at least one of HT-29(ATCC Accession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCCAccession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), or BXPC-3 (ATCC Accession No. CRL-1687)cells and not to non-neoplastic cells.
 2. The purified polypeptide ofclaim 1, wherein said polypeptide binds to ASPC-1 (ATCC Accession No.CRL-1682) and BXPC-3 (ATCC Accession No. CRL-1687) cells and not tonon-neoplastic cells, and wherein said neoplastic cell is a stomachadenocarcinoma, colorectal adenocarcinoma, squamous cell lung carcinoma,lung adenocarcinoma, squamous cell carcinoma of the esophagus,adenocarcinoma of the pancreas, adenocarcinoma of the prostate, ductalcarcinoma of the breast, lobular carcinoma of the breast, adenocarcinomaof the ovary, or adenocarcinoma of the uterus cell.
 3. The purifiedpolypeptide of claim 1, wherein said polypeptide binds to HT-29 (ATCCAccession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCCAccession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), and BXPC-3 (ATCC Accession No. CRL-1687)cells and not to non-neoplastic cells, and wherein said neoplastic cellis a stomach adenocarcinoma, colorectal adenocarcinoma, squamous celllung carcinoma, lung adenocarcinoma, squamous cell carcinoma of theesophagus, adenocarcinoma of the pancreas, urothel carcinoma of theurinary bladder, renal cell carcinoma of the kidney, adenocarcinoma ofthe prostate, ductal carcinoma of the breast, lobular carcinoma of thebreast, adenocarcinoma of the ovary, or adenocarcinoma of the uteruscell.
 4. The purified polypeptide of claim 1, wherein said polypeptidebinds to CACO-2 (ATCC Accession No. HBT-37; DSMZ Accession No. ACC 169)and COLO-206F (DSMZ Accession No. ACC 21) cells and not tonon-neoplastic cells, and wherein said neoplastic cell is a colorectaladenocarcinoma or adenocarcinoma of the endometrium cell.
 5. A purifiedpolypeptide that induces apoptosis of a neoplastic cell to which itbinds, but does not induce apoptosis of a non-neoplastic cell, whereinsaid polypeptide specifically binds to a stomach adenocarcinoma,colorectal adenocarcinoma, squamous cell lung carcinoma, lungadenocarcinoma, squamous cell carcinoma of the esophagus, adenocarcinomaof the pancreas, urothel carcinoma of the urinary bladder, renal cellcarcinoma of the kidney, adenocarcinoma of the prostate, ductalcarcinoma of the breast, lobular carcinoma of the breast, adenocarcinomaof the ovary, adenocarcinoma of the endometrium, or adenocarcinoma ofthe uterus cell and not to a non-neoplastic cell.
 6. A purifiedpolypeptide that inhibits cell proliferation when bound to a neoplasticcell, but does not inhibit cell proliferation of a non-neoplastic cell,wherein said polypeptide specifically binds to at least one of HT-29(ATCC Accession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCCAccession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144), COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), or BXPC-3 (ATCC Accession No. CRL-1687)cells and not to non-neoplastic cells.
 7. The purified polypeptide ofclaim 6, wherein said polypeptide binds to ASPC-1 (ATCC Accession No.CRL-1682) and BXPC-3 (ATCC Accession No. CRL-1687) cells and not tonon-neoplastic cells, and wherein said neoplastic cell is a stomachadenocarcinoma, colorectal adenocarcinoma, squamous cell lung carcinoma,lung adenocarcinoma, squamous cell carcinoma of the esophagus,adenocarcinoma of the pancreas, adenocarcinoma of the prostate, ductalcarcinoma of the breast, lobular carcinoma of the breast, adenocarcinomaof the ovary, or adenocarcinoma of the uterus cell.
 8. The purifiedpolypeptide of claim 6, wherein said polypeptide binds to HT-29 (ATCCAccession No. HTB-38; DSMZ Accession No. ACC 299), CACO-2 (ATCCAccession No. HBT-37; DSMZ Accession No. ACC 169), COLO-320 (DSMZAccession No. ACC 144). COLO-206F (DSMZ Accession No. ACC 21), ASPC-1(ATCC Accession No. CRL-1682), and BXPC-3 (ATCC Accession No. CRL-1687)cells and not to non-neoplastic cells, and wherein said neoplastic cellis a stomach adenocarcinoma, colorectal adenocarcinoma, squamous celllung carcinoma, lung adenocarcinoma, squamous cell carcinoma of theesophagus, adenocarcinoma of the pancreas, urothel carcinoma of theurinary bladder, renal cell carcinoma of the kidney, adenocarcinoma ofthe prostate, ductal carcinoma of the breast, lobular carcinoma of thebreast, adenocarcinoma of the ovary, or adenocarcinoma of the uteruscell. 9.-21. (canceled)
 22. The purified polypeptide of claim 1, whereinsaid polypeptide comprises a sequence that is substantially identical tothe amino acid sequence of SEQ ID NO:1.
 23. The purified polypeptide ofclaim 1, wherein said polypeptide comprises a sequence that issubstantially identical to the amino acid sequence of SEQ ID NO:3. 24.The purified polypeptide of claim 1, wherein said polypeptide comprisesa sequence that is substantially identical to the amino acid sequence ofSEQ ID NO:5.
 25. The purified polypeptide of claim 1, wherein saidpolypeptide comprises a sequence that is substantially identical to theamino acid sequence of SEQ ID NO:7.
 26. The purified polypeptide ofclaim 1, wherein said polypeptide comprises a sequence that issubstantially identical to the amino acid sequence of SEQ ID NO:9. 27.The purified polypeptide of claim 1, wherein said polypeptide comprisesa sequence that is substantially identical to the amino acid sequence ofSEQ ID NO:11.
 28. (canceled)
 29. A purified polypeptide comprising aminoacid 26-31, 49-51, and 88-95 of SEQ ID NO:1.
 30. (canceled)
 31. Apurified polypeptide comprising amino acids 11-18, 36-43, and 82-90 ofSEQ ID NO:3.
 32. (canceled)
 33. A purified polypeptide comprising aminoacids 26-34, 52-58, and 97-103 of SEQ ID NO:5.
 34. (canceled)
 35. Apurified polypeptide comprising amino acids 11-18, 36-43, and 82-100 ofSEQ ID NO:7.
 36. (canceled)
 37. A purified polypeptide comprising aminoacids 26-34, 51-54, and 91-99 of SEQ ID NO:9.
 38. (canceled)
 39. Apurified polypeptide comprising amino acids 16-22, 40-47, and 86-100 ofSEQ ID NO:11.
 40. (canceled)
 41. A purified polypeptide comprising aminoacid 26-31, 49-51, and 88-95 of SEQ ID NO:1 and amino acids 11-18,36-43, and 82-90 of SEQ ID NO:3.
 42. (canceled)
 43. A purifiedpolypeptide comprising amino acids 26-34, 52-58, and 97-103 of SEQ IDNO:5 and amino acids 11-18, 36-43, and 82-100 of SEQ ID NO:7. 44.(canceled)
 45. A purified polypeptide comprising amino acids 26-34,51-54, and 91-99 of SEQ ID NO:9 and amino acids 16-22, 40-47, and 86-100of SEQ ID NO:11.
 46. The purified polypeptide of claim 1, wherein saidpolypeptide is a monoclonal antibody.
 47. The purified polypeptide ofclaim 46, wherein said monoclonal antibody is a human monoclonalantibody. 48-76. (canceled)
 77. Use of the purified polypeptide of claim1, in a method of treating a proliferative disorder in a mammal, saidmethod comprising the step of contacting a cell or tissue sample withthe purified polypeptide of claim 1, wherein binding of said purifiedpolypeptide to said cell or tissue sample results in the induction ofapoptosis of said cell or tissue sample. 78-83. (canceled)
 84. Use ofthe purified polypeptide of claim 1, in a method of treating aproliferative disorder in a mammal, said method comprising the step ofcontacting a cell or tissue sample with the purified polypeptide ofclaim 1, wherein binding of said purified polypeptide to said cell ortissue sample results in a reduction in proliferation of said cell or ofa cell in said tissue sample. 85-110. (canceled)